Vinca derivatives

ABSTRACT

The present invention relates to derivatives of vinca alkaloids. Pharmaceutical compositions containing these compounds as well as processes of preparation and treatment of various conditions are also disclosed.

The present invention claims benefit of U.S. Provisional PatentApplication Ser. No. 60/526,912, filed Dec. 4, 2003.

FIELD OF THE INVENTION

The present invention relates to derivatives of the vinca alkaloidswhich are potent inhibitors of cellular mitosis and proliferation, aswell as pharmaceutical compositions, preparation processes, and methodsof use for treatment of various conditions.

BACKGROUND OF THE INVENTION

Cellular Proliferation and Cancer

The disruption of external or internal regulation of cellular growth canlead to uncontrolled cellular proliferation and in cancer, tumorformation. This loss of cellular growth control can occur at many levelsand, indeed, does occur at multiple levels in most tumors. Under thesecircumstances, although tumor cells can no longer control their ownproliferation, such cells still must use the same basic cellularmachinery employed by normal cells to drive their growth andreplication.

Mitosis and Spindle Formation

In a process known as mitosis, cancer cells, like all mammalian cells,multiply through replication and segregation of the originalchromosomes. Following DNA replication in the S phase, the cellsprogress in the G2 phase. During the G2 phase, cells continue toincrease in mass and prepare for mitosis. If chromosome damage ispresent in the G2 phase, the affected cell responds by activating the G2phase checkpoint, which prevents progression into mitosis. In theabsence of DNA damage or following repair of damage, the G2 phase cellsthen enter the M phase in which the identical pairs of chromosomes areseparated and transported to opposite ends of the cell. The cell thenundergoes division into two identical daughter cells.

In a process known as spindle formation, the cell utilizes the mitoticspindle apparatus to separate and pull apart the chromosomes. Thisapparatus, in part, consists of a network of microtubules that formduring the first stage of mitosis. Microtubules are hollow tubes thatare formed by the assembly of tubulin heterodimers from alpha- andbeta-tubulin. The assembly of tubulin into microtubules is a dynamicprocess with tubulin molecules being constantly added and subtractedfrom each end.

Vinca Compounds as Inhibitors of Mitosis and Cellular Proliferation

In general, vinca compounds are known to be inhibitors of mitosis andcellular proliferation. In particular, the antiproliferative activity ofthe vinca alkaloid class of drugs has been shown to be due to theirability to bind tubulin. Assembly of tubulin into microtubules isessential for mitosis and the binding of the vincas to tubulin leads tocell cycle arrest in M phase and subsequently to apoptosis. For example,at low concentrations, these compounds interfere with the dynamics ofmicrotubule formation. At higher concentrations, they cause microtubuledisassembly, and at still higher concentrations, the formation oftubulin paracrystals.

Moreover, the anti-cancer activity of vinca alkaloids is generallybelieved to result from a disruption of microtubules resulting inmitotic arrest. However, cytotoxicity of vinca alkaloids also has beendemonstrated in non-mitotic cells. Considering the role of microtubulesin many cellular processes, the cytotoxic action of vinca alkaloids mayinvolve contributions from inhibition of non-mitoticmicrotubule-dependent processes.

Cytotoxicity may also be a consequence of changes in membrane structureresulting from the partitioning of vinca alkaloids into the lipidbilayer. Studies with another tubulin binding compound, taxol, haveshown that cell cycle arrest was not a precondition for apoptosis byagents of this type. Therefore, the anti-cancer activity of vincaalkaloids may be the result of disruption of a number of distinctmicrotubule-dependent and possibly microtubule-independent processes.

The assembly of tubulin into microtubules is a complex process involvingdynamic instability (i.e. the switching between periods of slow growthand rapid shortening at both ends of the microtubule), and treadmilling(i.e. the addition of tubulin to one end of the microtubule occurring atthe same rate as loss of tubulin from the other). Low concentrations ofvinca alkaloids have been shown to bind to the ends of the microtubulesand suppress both microtubule instability and treadmilling during themetaphase stage of mitosis. For example, vinca alkaloids have been shownto stabilize microtubule plus ends and destabilize microtubule minusends. Although the spindle is retained under these conditions, there isfrequently abnormal alignment of condensed chromosomes. At higherconcentrations of vinca alkaloids, the spindle is not present and thechromosome distribution resembles that of prometaphase cells. At bothlow and high concentrations of vincas, mitotic arrest results fromactivation of metaphase-anaphase checkpoint. The molecular basis of thischeckpoint is a negative signal sent from the kinetochore of chromosomesthat are not attached to microtubules. This signal prevents theactivation of pathways that result in the initiation of anaphase events.

Although there is a common binding site for the vinca alkaloids ontubulin, the members of this class do behave differently. The relativeoverall affinities for β-tubulin binding arevincristine>vinblastine>vinorelbine>vinflunine, but there is nosignificant difference in the affinity of all four drugs for tubulinheterodimers. The discrepancy has primarily been explained bydifferences in the affinities of vinca-bound heterodimers for spiralpolymers and the binding of drug to unliganded polymers. For example,tubulin spirals induced by vinflunine are significantly smaller thanthose induced by vinorelbine.

In addition, vinca alkaloids also differ in their effects on microtubuledynamics. Vinflunine and vinorelbine suppress dynamic instabilitythrough: slowing the microtubule growth rate, increasing the meanduration of a growth event and reducing the duration of shortening. Incontrast, vinblastine reduces the rate of shortening and increases thepercentage of time the microtubules spend in the attenuated state.Vinblastine, vinorelbine, and vinflunine all suppress treadmilling, withvinblastine displaying the greatest potency.

In Vivo Properties

The vinca derivatives fall into the general class of cytotoxicanti-cancer agents and, as such, suffer from the same problem as allcytotoxics—i.e., toxicity. Vincristine and vinblastine are neurotoxic.Vinorelbine, which is structurally very similar to vinblastine andvincristine and is only slightly less potent, is less neurotoxic. Thischange in toxicity cannot be explained by examination of the bindingaffinity of these compounds for tubulin alone. It has been postulated toarise from an increase in sensitivity to changes in microtubule dynamicsin tumor cells and, as described above, these compounds have been shownto have subtly different effects. It could also arise from changes incellular uptake of the drug. Vinflunine is not very potent in vitro yetis active in vivo, and this has been attributed to its superior cellularuptake. There are also quite significant differences in the profile ofefficacy of vinca alkaloids. Vincristine has found wide use in thetreatment of hematologic malignancies including leukemias and lymphomas.It is also widely used in pediatric solid tumors and, in the past, insmall cell lung cancer. Vinblastine is an important component of thecombination regimen that is curative for testicular cancer. Vinorelbineis quite different and has found use mainly in breast cancer andnon-small cell lung cancer.

There remains a need for novel vinca derivatives with improvedpharmacological and therapeutic properties, improved processes for thepreparations of such vinca derivative compounds, correspondingpharmaceutical compositions, and methods of use.

The present invention is directed to achieving these objectives.

SUMMARY OF THE INVENTION

The present invention relates to a compound of Formula (I) as follows:

where:

-   R₁ is:    -   alkyl;    -   alkenyl;    -   alkynyl;    -   aryl;    -   heterocyclyl;    -   halogen;    -   CN;    -   CH(O);    -   COR₅;    -   C(O)NHR₅;    -   C(O)N R₅R₆;    -   C(S)NH₂;    -   C(O)NHNH₂;    -   C(O)NR₅NH₂;    -   C(O)NR₅NHR₆;    -   C(O)NR₅NR₆R₇;    -   C(O)NHNHR₅;    -   C(O)NHNR₅R₆;    -   C(O)NHOH;    -   SO₂NHNH₂;    -   SO₂NR₅NH₂;    -   SO₂NR₅NHR₆;    -   SO₂NR₅NR₆R₇;    -   SO₂NHNHR₅;    -   SO₂NHNR₅R₆;    -   CO₂R₅;    -   SR₅;    -   SSR₅;    -   SO₂NHR₅;    -   SO₂NR₅R₆;    -   B(OR₅)₂;    -   CF₃;    -   SH;    -   SO₂NH₂;    -   NH₂;    -   NHR₅;    -   NHSO₂R₅;    -   NR₅R₆;    -   NHCOR₅;    -   NR₅COR₆;    -   NR₅SO₂R₆; or-   R₂=alkyl or CH(O);-   R₃=hydrogen, alkyl, or C(O)R₅;-   R₄=hydrogen or C(O)R₅;-   R₅, R₆ and R₇ each are independently alkyl, alkenyl, alkynyl, aryl,    or heterocyclyl;-   R₈=hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl,    acyl, or thioalkyl;-   R₉=OH and R₁₀=H or R₉ and R₁₀ together form a bridging double bond.-   R₅ and R₆ could form a ring as could R₆ and R₇.-   X=OR₅, NR₅R₆, NHNH₂, NHNHC(O)R₅, OH; NHR₅; NH₂; or NHNHC(O)H;-   R₄ and X may be linked together with intervening atoms to form a    ring; R₁ and R₈ may be linked together; or a pharmaceutically    acceptable salt thereof, wherein the alkyl and alkenyl groups may be    branched, straight, unsubstituted, and/or substituted and wherein    the aryl, alkynyl, and heterocyclyl groups are substituted or    unsubstituted, with the proviso that when R₈=H, R₉=OH, and R₁₀=H,    then R₁≠Br, I, OH, or OMe.    More preferably:-   R₉=OH, R₁₀=H as in vincristine and vinblastine-   R₃=Ac as in vincristine and vinblastine-   R₄=H as in vincristine and vinblastine-   X=Me as in vincristine and vinblastine-   R₁=alkyl;    -   alkenyl;    -   alkynyl;    -   aryl;    -   heterocyclyl;    -   halogen;    -   CN;    -   CH(O);    -   COR₅;    -   C(O)NHR₅    -   CO₂R₅;    -   SR₅;    -   SSR₅;    -   SH;    -   NH₂;    -   NHR₅;    -   NR₅R₆;        Most preferably:-   R₁=alkyl;    -   alkenyl;    -   alkynyl;    -   halogen;    -   CN;    -   SR₅;    -   SSR₅;    -   SH;    -   NH₂;    -   NHR₅;    -   NR₅R₆; where R₅ and R₆ form a ring

Another aspect of the present invention relates to a process forpreparation of a derivative product compound of Formula (I) as follows:

where:

-   R₁ is:    -   alkyl;    -   alkenyl;    -   alkynyl;    -   aryl;    -   heterocyclyl;    -   CN;    -   CH(O);    -   COR₅;    -   C(O)NR₅R₆;    -   C(O)NHR₅;    -   C(O)NH₂;    -   C(O)NHNH₂;    -   C(O)NR₅NH₂;    -   C(O)NR₅NHR₆;    -   C(O)NR₅NR₆R₇;    -   C(O)NHNHR₅;    -   C(O)NHNR₅R₆;    -   C(O)NHOH;    -   SO₂NHNH₂;    -   SO₂NR₅NH₂;    -   SO₂NR₅NHR₆;    -   SO₂NR₅NR₆R₇;    -   SO₂NHNHR₅;    -   SO₂NHNR₅R₆;    -   CO₂R₅;    -   SR₅;    -   SSR₅;    -   SO₂NHR₅;    -   SO₂NR₅R₆;    -   B(OR₅)₂;    -   CF₃;    -   SH;    -   SO₂NH₂;    -   NH₂;    -   NHR₅;    -   NHCOR₅;    -   NHSO₂R₅;    -   NR₅R₆;    -   NHCOR₅;    -   NR₅COR₆; or    -   NR₅SO₂R₆;-   R₂=alkyl or CH(O);-   R₃=hydrogen, alkyl, or C(O)R₅;-   P₄=hydrogen or C(O)R₅;-   R₅, P₆ and R₇ each are independently alkyl, alkenyl, alkynyl, aryl,    or heterocyclyl;-   R₈=hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl,    acyl, or thioalkyl;-   R₉=OH and R₁₀=H or R₉ and R₁₀ together form a bridging double bond;-   R₅ and R₆ could form a ring or R₆ and R₇ could form a ring;-   X=OR₅, NR₅P₆, NHNH₂, NHNHC(O)R₅, OH; NHR₅; NH₂; or NHNHC(O)H;-   R₄ and X may be linked together with intervening atoms to form a    ring; R₁ and R₈ may be linked together; or a pharmaceutically    acceptable salt thereof, wherein the alkyl and alkenyl groups may be    branched, straight, unsubstituted, and/or substituted and wherein    the aryl, alkynyl, and heterocyclyl groups are substituted or    unsubstituted, with the proviso, that when R₈=H, R₉=OH, and R₁₀=H,    then R₁≠Br, I, OH, or OMe. The process also involves converting an    intermediate compound of formula:

-    wherein Y₁ is a halogen and Y₂ is halogen or hydrogen, under    conditions effective to produce the product compound of Formula (I).

Another aspect of the present invention relates to a process forpreparation of a derivative product compound of Formula (I) as follows:

where:

-   R₁ is:    -   halogen;-   R₂=alkyl or CH(O);-   R₃=hydrogen, alkyl, or C(O)R₅;-   R₄=hydrogen or C(O)R₅;-   R₅, R₆ and R₇ each are independently alkyl, alkenyl, alkynyl, aryl,    or heterocyclyl;-   R₈=hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl,    acyl, or thioalkyl;-   R₉=OH and R₁₀=H or R₉ and R₁₀ together form a bridging double bond;-   R₅ and R₆ could form a ring or R₆ and R₇ could form a ring;-   X=OR₅, NR₅R₆, NHNH₂, NHNHC(O)R₅, OH; NHR₅; NH₂; or NHNHC(O)H;-   R₄ and X may be linked together with intervening atoms to form a    ring; R₁ and R₈ may be linked together; or a pharmaceutically    acceptable salt thereof, wherein the alkyl and alkenyl groups may be    branched, straight, unsubstituted, and/or substituted and wherein    the aryl, alkynyl, and heterocyclyl groups are substituted or    unsubstituted, with the proviso, that when R₈=H, R₉=OH, and R₁₀=H,    then R₁≠Br, I, OH, or OMe. The process also involves halogenating a    starting material of the formula:

-    under conditions effective to form the derivative product compound.

The present invention also relates to a method for inhibiting cellproliferation in mammals, which comprises administering atherapeutically effective amount of the compound of Formula (I) to themammal.

The present invention also relates to a method for treating a conditionin mammals, which comprises administering a therapeutically effectiveamount of the compound of Formula (I) to the mammal. The condition canbe bacterial infection, allergy, heart disease, AIDS, HumanT-lymphotropic virus 1 infection, Human herpesvirus 3, Human herpesvirus4, Human papillomavirus, diabetes mellitus, rheumatoid arthritis,Alzheimer's Disease, inflammation, arthritis, asthma, malaria,autoimmune disease, eczema, Lupus erythematosus, psoriasis, rheumaticdiseases, Sjogren's syndrome, and viral infection.

The present invention also relates to a pharmaceutical composition ofmatter, which comprises the compound of Formula (I) and one or morepharmaceutical excipients.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel derivatives of the vincaalkaloids, corresponding pharmaceutical compositions, preparationprocesses, and methods of use for treatment of various diseases.

In general, the novel compounds of the vinca family of compounds of thepresent invention, include derivatives of vincristine, vinblastine,anhydrovinblastine, and anhydrovincristine, etc. In accordance with thepresent invention, such derivative compounds are represented by thechemical structure of Formula (I) as shown herein.

In particular, the present invention relates to a compound of Formula(I) as follows:

where:

-   R₁=alkyl;    -   alkenyl;    -   alkynyl;    -   aryl;    -   heterocyclyl;    -   halogen;    -   CN;    -   CH(O);    -   COR₅;    -   C(O)NR₅R₆;    -   C(O)NHR₅;    -   C(O)NH₂;    -   C(O)NHNH₂;    -   C(O)NR₅NH₂;    -   C(O)NR₅NHR₆;    -   C(O)NR₅NR₆R₇;    -   C(O)NHNHR₅;    -   C(O)NHNR₅R₆;    -   C(O)NHOH;    -   SO₂NHNH₂;    -   SO₂NR₅NH₂;    -   SO₂NR₅NHR₆;    -   SO₂NR₅NR₆R₇;    -   SO₂NHNHR₅;    -   SO₂NHNR₅R₆;    -   CO₂R₅;    -   SR₅;    -   SSR₅;    -   SO₂NHR₅;    -   SO₂NR₅R₆;    -   B(OR₅)₂;    -   CF₃;    -   SH;    -   SO₂NH₂;    -   NH₂;    -   NHR₅;    -   NHSO₂R₅;    -   NR₅R₆;    -   NHCOR₅;    -   NR₅COR₆; or    -   NR₅SO₂R₆;-   R₂=alkyl or CH(O);-   R₃=hydrogen, alkyl, or C(O)R₅;-   R₄=hydrogen or C(O)R₅;-   R₅, R₆ and R₇ each are independently alkyl, alkenyl, alkynyl, aryl,    or heterocyclyl;-   R₈=hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl,    acyl, or thioalkyl;-   R₉=OH and R₁₀=H or R₉ and R₁₀ together form a bridging double bond;-   R₅ and R₆ could form a ring or R₆ and R₇ could form a ring;-   X=OR₅, NR₅R₆, NHNH₂, NHNHC(O)R₅, OH; NHR₅; NH₂; or NHNHC(O)H;-   R₄ and X may be linked together with intervening atoms to form a    ring; or a pharmaceutically acceptable salt thereof, wherein the    alkyl and alkenyl groups may be branched, straight, unsubstituted,    and/or substituted and wherein the aryl, alkynyl, and heterocyclyl    groups are substituted or unsubstituted, with the proviso, that when    R₈=H, R₉=OH, and R₁₀=H, then R₁≠Br, I, OH, or OMe.

In one embodiment, the present invention relates to a compound whereR₃=acetyl.

In another embodiment, the present invention relates to a compound whereR₄=hydrogen.

In another embodiment, the present invention relates to a compound whereX=OMe.

In another embodiment, the present invention relates to a compound whereR₃=acetyl, R₄=hydrogen, and X=OMe.

In another embodiment, the present invention relates to a compound whereR₂=CH(O).

In another embodiment, the present invention relates to a compound whereR₂=alkyl.

Representative examples of the compounds of Formula (I) are set forth inTable 1 below

TABLE 1 Compounds of Formula (I) Example NAME OF VINCA Number COMPOUNDOF FORMULA (I) COMPOUND 5

12′-phenylvincristine 6

12′-phenylvinblastine 7

12′-(4-methoxyphenyl)vincristine 8

12′-(4-methoxyphenyl)vinblastine 9

12′-(3-methoxyphenyl)vinblastine 10

12′-(4-fluorophenyl)vinblastine 11

12′-(3-fluorophenyl)vinblastine 12

12′-(3-hydroxyphenyl)vinblastine 13

12′-(3-pyridyl)vinblastine 14

12′-(3-thienyl)vinblastine 15

12′-(2-thiazolyl)vinblastine 16

12′-(trimethylsilylethynyl)vinblastine 17

12′-ethynylvinblastine 18

12′-propynylvinblastine 19

12′-(2-phenylethynyl)vinblastine 20

12′-(3-methylbutynyl)vinblastine 21

12′-(3-methylbutynyl)vincristine 22

12′-hexynylvincristine 23

12′-hexynylvinblastine 24

12′-(N,N-dimethylaminopropynyl)vinblastine 25

12′-vinylvinblastine 26

12′-(2-ethoxycarbonylvinyl)vinblastine 27

12′-(2-tert-butoxycarbonylvinyl)vinblastine 28

12′-(2-carboxylvinyl)vinblastine 29

12′-(3-oxohex-1-enyl)vinblastine 30

12′-(2-cyanovinyl)vinblastine 31

12′-(3-tert-butoxycarbonylaminopropenyl)vinblastine 32

12′-(4-hydroxybutylsulfanyl)vinblastine 33

12′-(4-hydroxypropylsulfanyl)vinblastine 34

12′-(4-methanesulfonyloxypropylsulfanyl)vinblastine 35

12′-(2-hydroxyethylsulfanyl)vinblastine 36

12′-(4-methoxybenzylsulfanyl)vinblastine 37

12′-(2-chlorobenzylsulfanyl)vincristine 38

12′-(2-fluorobenzylsulfanyl)vincristine 39

12′-(propylsulfanyl)vinblastine 40

12′-(ethylsulfanyl)vincristine 41

12′-(ethylsulfanyl)vinblastine 42

12′-(methylsulfanyl)vincristine 43

12′-(methylsulfanyl)vinblastine 44

12′-(tert-butoxycarbonylmethylsulfanyl)vinblastine 45

12′-(carboxymethylsulfanyl)vinblastine 46

12′-(methylaminocarbonylmethylsulfanyl)vinblastine 47

12′-(methoxycarbonylethylsulfanyl)vincristine 48

12′-(2-(N,N-dimethylamino)ethylsulfanyl)vinblastine 49

12′-(3-(morpholin-4-yl)propylsulfanyl)vinblastine 50

12′-(3-(piperidin-1-yl)propylsulfanyl)vinblastine 51

12′-[2-Pyrrolidin-1-yl-ethylsulfanyl]vinblastine 52

12′-(2-(acetylamino)ethylsulfanyl)vinblastine 53

12′-thiovinblastine 54

12′-(3-hydroxyphenylsulfanyl)vincristine 55

12′-(2-hydroxyphenylsulfanyl)vinblastine 56

12′-(2-chlorophenylsulfanyl)vincristine 57

12′-(methyldisulfanyl)vinblastine 58

12′-(isopropyldisulfanyl)vinblastine 59

12′-(tert-butyldisulfanyl)vinblastine 60

di(12′-vinblastine)disulfide 61

12′-formylvinblastine 62

12′-formylvincristine 63

12′-(hydroxymethyl)vinblastine 64

12′-(N-isopropylaminomethyl)vinblastine 65

12′-cyanovinblastine 66

12′-cyanovincristine 67

12′-(methycarbonyl)vinblastine 68

12′-(2,2,2-trichloroethylcarbonyl)vinblastine 69

12′-N-(methylaminocarbonyl)vinblastine 70

12′-acetylvinblastine 71

12′-(3-methylbutanoyl)vincristine 72

12′-hexanoylvincristine 73

12′-(3-methylbutyl)vincristine 74

12′-hexylvincristine 75

12′-methylvinblastine 76

12′-methylvincristine 77

12′-ethylvinblastine 78

12′-ethylvincristine 79

12′-(N-methyl-N-phenylamino)vinblastine 80

12′-aminovinblastine 81

12′-(N,N-dimethylamino)vinblastine 82

12′-(phenylamino)vinblastine 83

12′-(4-methoxyphenylamino)vinblastine 84

12′-(4-trifluoromethylphenylamino)vinblastine 85

12′-(1-piperidinyl)vinblastine 86

12′-(4-morpholino)vinblastine 87

12′-(pyrrolidin-1-yl)vinblastine 88

12′-(azetidin-1-yl)vinblastine 89

12′-(3-methylpyrazol-1-yl)vinblastine 90

12′,13′-diiodovincristine 91

12′,13′-diiodovinblastine 92

13′-iodo-12′-methylvincristine 93

12′,13′-dimethylvincristine 94

13′-ethyl-12′-methylvincristine 95

12′,13′-diethylvincristine 96

13′-acetyl-12′-methylvincristine 97

12′-bromoanhydrovinblastine 98

12′-iodoanhydrovinblastine 99

12′-bromoanhydrovincristine

In yet another embodiment of the present invention, a complex can beformed which includes 2 structures of Formula (I) joined together attheir R₁ groups, where each R₁ is —S—.

The synthetic reaction scheme for the preparation of compounds of (I) isdepicted below.

A synthetic scheme for preparing compounds of Formula (I) is shown inScheme 1 below. A vinca alkaloid is treated with eitherN-bromosuccinimide or N-iodosuccinimide to introduce halogens in the 12′and 13′-positions. Pd-mediated coupling is then used to introduce otherfunctionality at these position. This methodology can be used tointroduce alkyl, alkenyl, alkynyl, aryl, heterocyclyl, acyl, cyano,amino, and formyl groups and to form sulphides. Each of these groups canthen be subjected to further derivatization following standard methodsof organic synthesis.

In practicing the above process, a variety of catalysts may be utilized,such as palladium chloride, palladium acetate,tris(dibenzylideneacetone)palladium(0),tetrakis(triphenylphosphine)palladium(0),dichlorobis(triphenylphosphine)palladium(II),benzylchlorobis(triphenylphosphine)palladium(II), or[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).

Based on the results obtained in the standard pharmacological testprocedures described below, the compounds of the present invention areuseful in inhibiting cellular proliferation in a mammal by administeringto such mammal an effective amount of compound(s) of the presentinvention.

In particular, such vinca compound derivatives are useful asantineoplastic agents. More particularly, the compounds of the presentinvention are useful for inhibiting the growth of neoplastic cells,causing cell death of neoplastic cells, and eradicating neoplasticcells. The compounds of the present invention are, therefore, useful fortreating solid tumors, (e.g., sarcomas), carcinomas, (e.g.,astrocytomas), lymphomas, (e.g., adult T-cell lymphoma), differentcancer disease types, (e.g., prostate cancer, breast cancer, small celllung cancer, ovarian cancer), Hodgkin's Disease, and other neoplasticdisease states (e.g., leukemias, particularly adult T-cell leukemias).

Since vinca compounds are known to be tubulin inhibitors, the compoundsof the present invention would also be expected to be useful in treatingthe following conditions: bacterial infection; allergy; heart disease;AIDS; Human T-lymphotropic virus 1 infection; Human herpesvirus 3; Humanherpesvirus 4; Human papillomavirus; diabetes mellitus; rheumatoidarthritis; Alzheimer's Disease; inflammation; arthritis; asthma;malaria; autoimmune disease; eczema; Lupus erythematosus; psoriasis;rheumatic diseases; Sjogren's syndrome; and viral infection.

The vinca derivative compounds of the present invention can beadministered alone, as indicated above, or utilized as biologicallyactive components in pharmaceutical compositions with suitablepharmaceutically acceptable carriers, adjuvants and/or excipients.

In accordance with the present invention, the compounds and/orcorresponding compositions can be introduced via differentadministration routes, which include orally, parenterally,intravenously, intraperitoneally, by intranasal instillation, or byapplication to mucous membranes, such as, that of the nose, throat, andbronchial tubes.

The active compounds of the present invention may be orallyadministered, for example, with an inert diluent, or with an assimilableedible carrier, or they may be enclosed in hard or soft shell capsules,or they may be compressed into tablets.

The quantity of the compound administered will vary depending on thepatient and the mode of administration and can be any effective amount.The quantity of the compound administered may vary over a wide range toprovide in a unit dosage an effective amount of from about 0.01 to 20mg/kg of body weight of the patient per day to achieve the desiredeffect. The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained. Preferredcompositions according to the present invention are prepared so that anoral dosage unit contains between about 1 and 250 mg of active compound.

For example, with oral therapeutic administration, these activecompounds may be incorporated with excipients and used in the form oftablets, capsules, elixirs, suspensions, syrups, and the like. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compound in these compositions may, ofcourse, be varied and may conveniently be between about 2% to about 60%of the weight of the unit.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both.

These active compounds and/or pharmaceutical compositions may also beadministered parenterally. Solutions of these active compounds and/orcompositions can be prepared in water. Dispersions can also be preparedin glycerol, liquid polyethylene glycols, and mixtures thereof in oils.

Illustrative oils are those of animal, vegetable, or synthetic origin,for example, peanut oil, soybean oil, or mineral oil. In general, water,saline, aqueous dextrose and related sugar solution, and glycols suchas, propylene glycol or polyethylene glycol, are preferred liquidcarriers, particularly for injectable solutions. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the pharmaceutical form of the presentinvention must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), suitable mixtures thereof, and vegetable oils.

The compounds and/or pharmaceutical compositions of the presentinvention may also be administered directly to the airways in the formof an aerosol. For use as aerosols, the compounds of the presentinvention in solution or suspension may be packaged in a pressurizedaerosol container together with suitable propellants, for example,hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. The materials of the present invention also maybe administered in a non-pressurized form such as in a nebulizer oratomizer.

Some of the compounds of the present invention can be in the form ofpharmaceutically acceptable acid-addition and/or base salts. All ofthese forms of salts are within the scope of the present invention.

Pharmaceutically acceptable acid addition salts of the compounds of thepresent invention include salts derived from nontoxic inorganic acids,such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid,hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphorous acid,and the like, as well as the salts derived from nontoxic organic acids,such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromaticacids, aliphatic and aromatic sulfonic acids, etc. Such salts thusinclude sulfates, pyrosulfates, bisulfates, sulfites, bisulfites,nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates,trifluoroacetates, propionates, caprylates, isobutyrates, oxalates,malonates, succinate suberates, sebacates, fumarates, maleates,mandelates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates,phenylacetates, citrates, lactates, malates, tartrates,methanesulfonates, and the like. Also contemplated are salts of aminoacids, such as arginates, gluconates, and galacturonates (see, forexample, Berge S. M. et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Science, 66:1-19 (1997), which is hereby incorporated byreference in its entirety).

The acid addition salts of said basic compounds are prepared bycontacting the free base forms with a sufficient amount of the desiredacid to produce the salt in the conventional manner.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenedianline, N-methylglucamine, and procaine(see, for example, Berge S. M. et al., “Pharmaceutical Salts,” Journalof Pharmaceutical Science, 66:1-19 (1997), which is hereby incorporatedby reference in its entirety).

The base addition salts of the acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional mariner.

Certain of the compounds of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms, including hydrated forms, are equivalent tounsolvated forms and are intended to be encompassed within the scope ofthe present invention.

The present invention can be used in conjunction with other know cancertreatments, including other chemotherapeutic agents and radiation.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are no means intended to limit its scope.

Spectroscopic analysis of products described in the experimentalprocedures below were performed with conventional or standard scientificinstrumentation known in the art. Proton NMR spectra were obtained on aBruker AC 300 spectrometer at 300 MHz or a Bruker 500 MHz spectrometerat 500 MHz and were referenced to tetramethylsilane as an internalstandard. Mass spectra were obtained on either a Shimadzu QP-5000 or aPE Sciex API 150 Mass Spectrometer.

Example 1 Preparation of 12′-Bromovinblastine Trifluoroacetate

A solution of vinblastine sulfate (0.5 g, 0.55 mmol) in trifluoroaceticacid (50 mL) under nitrogen was stirred at room temperature for 20 min.The flask was wrapped with foil to keep the reaction mixture in the darkand a solution of N-bromosuccinimide (103 mg, 0.58 mmol) intrifluoroacetic acid (25 mL) was added dropwise. After stirring for 18h, the reaction mixture was concentrated under reduced pressure, dilutedwith dichloromethane, and poured into ice water. The aqueous layer waswashed 3× with dichloromethane. The pH of the mixture was adjusted to11-12 with 3% NH₃ (aq). The organic layer was separated and the aqueouslayer was extracted with dichloromethane (3×) and the combined organiclayers were washed with water, dried (Na₂SO₄), and concentrated underreduced pressure. Purification by column chromatography (silica(deactivated by eluting with 10% triethylamine in hexane), EtOAc) gave amixture of mono and dibromides (0.33 g, 65%). Purification by reversedphase chromatography (C18, acetonitrile/water, 0.05% trifluoroaceticacid) gave 12′-bromovinblastine as a trifluoroacetate (0.42 g, 69%). ¹HNMR (300 MHz, CDCl₃) δ 8.09 (s, 1H), 7.62 (d, J=2 Hz, 1H), 7.21 (dd,J=9, 2 Hz, 1H), 6.97 (d, J=9 Hz, 1H), 6.52 (s, 1H), 6.10 (s, 1H), 5.85(dd, J=9, 4 Hz, 1H), 5.46 (s, 1H), 5.30 (d, J=9 Hz, 1H), 3.89 (m, 2H),3.80 (s, 6H), 3.73 (s, 1H), 3.65 (m, 1H), 3.63 (s, 3H), 3.50-2.90 (m,6H), 2.85 (m, 3H), 2.71 (s, 3H), 2.62 (s, 1H), 2.53-2.38 (m, 2H),2.36-2.10 (m, 2H), 2.11 (s, 3H), 1.90-1.70 (m, 3H), 1.54-1.25 (m, 7H),0.89 (t, J=7 Hz, 3H), 0.77 (t, J=7 Hz, 3H); ESI MS m/z 889, 891 [M+H]⁺.

Example 2 Preparation of 12′-Iodovinblastine

A solution of N-iodosuccinimide (254 mg, 1.13 mmol) in trifluoroaceticacid/methylene chloride (1:1, 16 mL) was cooled to approximately 0° C.in an ice-water jacketed addition funnel then added dropwise tovinblastine hydrogensulfate (1.08 g, 1.19 mmol) in trifluoroaceticacid/methylene chloride (1:1, 32 mL) at −15° C. The temperature wasmonitored by an internal thermometer and maintained at −15±3° C. duringthe course of the addition (45 min). After the addition was complete,the reaction mixture was stirred 10 min then poured carefully into arapidly stirring mixture of 10% sodium sulfite/satd sodiumhydrogencarbonate/chloroform (1:2:2, 200 mL). Solid sodiumhydrogencarbonate was then added in small portions until gas evolutionstopped. The solution was then extracted with chloroform (3×50 mL) andthe combined organic extracts were washed with 10% sodium sulfite (50mL) and brine (50 mL) and dried over magnesium sulfate. The solvent wasremove in vacuo to provide 12′-iodovinblastine (1.19 g, quantitative) asa tan foam which was carried forward without further purification: ¹HNMR (500 MHz, CD₃OD) δ 7.75 (d, J=1.1 Hz, 1H), 7.32 (dd, J=8.4, 1.4 Hz,1H), 7.00 (d, J=8.5 Hz, 1H), 6.56 (s, 1H), 6.31 (s, 1H), 5.83 (dd,J=10.2, 3.9 Hz, 1H), 5.36 (s, 1H), 5.29 (d, J=10.2 Hz, 1H), 3.91-4.07(m, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.64 (s, 3H), 3.58 (s, 1H), 3.36(d, J=14.3 Hz, 1H), 3.30 (m, 1H), 3.15-3.27 (m, 3H), 2.94 (dd, J=14.8,4.0 Hz, 1H), 2.71-2.85 (m, 4H), 2.71 (s, 3H), 2.41-2.49 (m, 2H), 2.26(dd, J=15.6 3.6 Hz, 1H), 2.07 (m, 1H), 2.02 (s, 3H), 1.87 (m, 1H), 1.66(m, 1H), 1.50 (d, J=14.1 Hz, 1H), 1.41 (m, 2H), 1.32 (q, J=7.4 Hz, 2H),0.90 (t, J=7.5 Hz, 3H), 0.81 (m, 1H), 0.75 (t, J=7.3 Hz, 3H); ESI MS m/z937 [M+H]+.

Example 3 Preparation of 12′-Bromovincristine

12′-Bromovincristine was prepared from vincristine following theprocedure described in Example 1, yield (1.41 g, 72%). ¹H NMR (500 MHz,DMSO-d₆) δ 11.16 (br s, 1H), 10.63 (br s, 1H), 9.38 (br s, 1H), 7.83 (d,J=1 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J=9 Hz, 1H), 7.23 (dd, J=9, 1 Hz,1H), 7.07 (s, 1H), 5.90 (dd, J=11, 6 Hz, 1H), 5.62 (d, J=10 Hz, 1H),5.18 (br s, 1H), 5.04 (s, 1H), 4.60 (s, 1H), 4.40 (m, 1H), 4.10-3.32(m), 3.88 (s, 3H), 3.67 (s, 3H), 3.55 (s, 3H), 3.27 (br d, J=15 Hz, 1H),3.12 (m, 3H), 3.02 (m, 1H), 2.76 (m, 1H), 2.30 (m, 1H), 2.21 (m, 1H),2.03 (s, 3H), 1.82 (m, 1H), 1.58-1.37 (m, 7H), 1.10 (m, 1H), 0.87 (t,J=7 Hz, 3H), 0.65 (t, J=7 Hz, 3H); ESI MS m/z 903, 905 [M+H]⁺.

Example 4 Preparation of 12′-Iodovincristine

A solution of N-iodosuccinimide (160 mg, 0.711 mmol) in trifluoroaceticacid/methylene chloride (1:1, 20 mL) was cooled to approximately 0° C.in an ice-water jacketed addition funnel then added dropwise tovinblastine hydrogensulfate (725 mg, 0.785 mmol) in trifluoroaceticacid/methylene chloride (1:1, 30 mL) at −15° C. The temperature wasmonitored by an internal thermometer and maintained at −15±3° C. duringthe course of the addition (30 min). The reaction was >97% complete asjudged by HPLC (C18, acetonitrile, H₂O, 0.5% trifluoroacetic acid).Additional N-iodosuccinimide (8.0 mg, 0.036 mmol) was added and stirredfor 10 minutes. HPLC indicated no remaining starting material. Thereaction mixture was treated with saturated aqueous sodium bicarbonate,and further neutralized with NaOH (3 N) until a pH of 8 was obtained.The solution was diluted with methylene chloride (150 mL). The organiclayer was washed with water and brine then dried over Na₂SO₄. Thesolvent was removed in vacuo to provide 12′-iodovincristine (0.68 g,91%) as a tan powder which was carried forward without furtherpurification: ¹H NMR (500 MHz, CD₃OD) δ 8.93 (s, 1H), 7.77 (d, J=1.0 Hz,1H), 7.32 (dd, J=8.5, 1.5 Hz, 1 H), 7.21 (s, 1H), 7.08 (d, J=8.5 Hz,1H), 6.86 (s, 1H), 5.83 (dd, J=10.0, 5.0 Hz, 1H), 5.40 (d, J=10.0 Hz,1H), 5.14 (s, 1H), 4.58 (s, 1H), 4.09-4.01 (m, 2H), 3.90 (s, 3H), 3.68(s, 3H), 3.62 (s, 3H), 3.39 (d, J=14.5 Hz, 1H), 3.35-3.22 (m, 4H), 3.04(s, 1H), 3.01 (m, 1H), 2.88-2.78 (m, 4H), 2.62 (m, 1H), 2.47 (dd,J=14.5, 4.0 Hz, 1H), 2.32 (dd, J=14.5 4.0 Hz, 1H), 2.08 (m, 1H), 2.00(s, 3H), 1.76 (m, 1H), 1.54-1.28 (m, 6H), 0.90 (t, J=7.5 Hz, 3H), 0.75(t, J=7.0 Hz, 3H); ESI MS m/z 937 [M+H]⁺.

Example 5 Preparation of 12′-Phenylvincristine

A mixture of 12′-bromovincristine (231 mg, 0.256 mmol), cesium carbonate(0.5 g, 1.5 mmol) and phenyl boronic acid (62.5 mg, 0.51 mmol) in1,4-dioxane (10 mL) was deoxygenated with argon.[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (21 mg,0.026 mmol) was added and the mixture again deoxygenated with an argonpurge, then heated at 60° C. for 24 h. After cooling, the reactionmixture was filtered through a short silica column, washing withdichloromethane then EtOAc, and filtrate concentrated under reducedpressure. Purification of the residue by flash chromatography (silica,EtOAc, then 99:1 EtOAc/EtOH) gave 12′-phenylvincristine (185 mg, 80%).¹H NMR (300 MHz, CDCl₃)δ 9.44 (br s, 1H), 8.77 (s, 1H), 8.21 (s, 1H),8.17 (br s, 1H), 7.71 (s, 1H), 7.63 (d, J=8 Hz, 2H), 7.47 (t, J=8 Hz,1H), 7.43 (d, J=8 Hz, 1H), 7.35 (t, J=7 Hz, 1H), 7.25 (d, J=8 Hz, 1H),6.96 (s, 1H), 6.80 (s, 1H), 5.83 (m, 1H), 5.42 (d, J=10 Hz, 1H), 5.24(d, J=10 Hz, 1H), 4.75 (s, 1H), 4.55 (s, 1H), 4.10-3.60 (m, 2H), 3.90(s, 1H), 3.89 (s, 3H), 3.79 (s, 1H), 3.73 (s, 3H), 3.68 (s, 3H),3.46-3.10 (m, 6H), 2.93 (m, 2H), 2.80 (s, 1H), 2.62 (m, 1H), 2.35 (m,2H), 2.07 (m, 4H), 1.82-1.55 (m, 3H), 1.50-1.16 (m, 6H), 0.89 (t, J=7Hz, 6H); ESI m/z 901 [M+H]⁺.

Example 6 Preparation of 12′-Phenylvinblastine

12′-Phenylvinblastine was prepared 12′-bromobinblastine and phenylboronic acid following the procedure described in Example 5, yield (45mg, 19%). ¹H NMR (300 MHz, CDCl₃) δ 9.89 (s, 1H), 8.08 (s, 1H), 7.69 (s,1H), 7.63 (d, J=8 Hz, 2H), 7.45 (d, J=7 Hz, 1H), 7.42 (t, J=8 Hz, 2H),7.34 (t, J=7 Hz, 1H), 7.19 (d, J=8 Hz, 1H), 6.66 (s, 1H), 6.11 (s, 1H),5.87 (dd, J=10, 4 Hz, 1H), 5.48 (s, 1H), 5.30 (d, J=10 Hz, 1H), 3.98 (t,J=13 Hz, 1H), 3.80 (s, 5H), 3.74 (s, 1H), 3.63 (s, 3H), 3.50-3.10 (m,6H), 2.90-2.75 (m, 2H), 2.72 (s, 3H), 2.72 (s, 1H), 2.53-2.37 (m, 2H),2.36-2.10 (m, 2H), 2.11 (s, 3H), 1.90-1.55 (m, 3H), 1.55-1.18 (m, 5H),2.11 (s, 3H), 0.90 (t, J=7 Hz, 3H), 0.85 (t, J=7 Hz, 3H); ESI MS m/z 887[M+H]⁺.

Example 7 Preparation of 12′-(4-Methoxyphenyl)vincristineTrifluoroacetate

The trifluoroacetate of 12′-(4-methoxyphenyl)vincristine was preparedfrom 12′-bromovincristine and 4-methoxyphenyl boronic acid following theprocedure described in Example 5. Purification by reversed phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid)gave 12′-(4-methoxyphenyl)vincristine trifluoroacetate (135 mg, 87%). ¹HNMR (300 MHz, CDCl₃) δ 8.76 (s, 1H), 8.21 (s, 1H), 8.16 (br s, 1H), 7.80(s, 1H), 7.53 (d, J=9 Hz, 2H), 7.44 (d, J=8 Hz, 1H), 7.19 (d, J=8 Hz,1H), 6.99 (d, J=8 Hz, 2H), 6.85 (s, 1H), 5.91 (m, 1H), 5.56 (m, 1H),5.19 (d, J=15 Hz, 1H), 4.83 (m, 1H), 4.59 (dd, J=15, 10 Hz, 1H),4.10-3.60 (m, 4H), 3.93 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H), 3.75 (s,3H), 3.70 (s, 1H), 3.60-3.00 (m, 8H), 3.11 (br s, 3H), 2.82 (m, 1H),2.56 (m, 1H), 2.41 (m, 1H), 2.07 (s, 3H), 1.96 (m, 1H), 1.67 (m, 2H),1.55 (q, J=7 Hz, 2H), 1.47-1.19 (m, 2H), 0.99 (t, J=7 Hz, 3H) 0.76 (m,3H); ESI m/z 931 [M+H]⁺.

Example 8 Preparation of 12′-(4-Methoxyphenyl)vinblastine

12′-(4-Methoxyphenyl)vinblastine was prepared from 12′-bromovinblastineand 4-methoxyphenyl boronic acid following the procedure described inExample 5 (39 mg, 26%). ¹H NMR (300 MHz, CDCl₃) δ 8.76 (s, 1H), 8.12 (s,1H), 7.55 (br s, 1H), 7.53 (d, J=9 Hz, 2H), 7.42 (dd, J=9, 2 Hz, 1H),7.14 (d, J=9 Hz, 1H), 6.99 (d, J=9 Hz, 2H), 6.53 (s, 1H), 6.13 (s, 1H),5.87 (dd, J=10, 5 Hz, 1H), 5.45 (d, J=10 Hz, 2H), 5.44 (s, 1H), 4.59 (m,1H), 4.00 (m, 1H), 3.94 (s, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.83 (s,3H), 3.70 (s, 3H), 3.60-2.80 (m, 12H), 2.71 (s, 3H), 2.63-2.40 (m, 2H),2.15 (m, 1H), 2.09 (s, 3H), 2.00-1.30 (m, 10H), 0.99 (t, J=7 Hz, 3H),0.73 (t, J=7 Hz, 3H); ESI m/z 917 [M+H]⁺.

Example 9 Preparation of 12′-(3-Methoxyphenyl)vinblastineTrifluoroacetate

To a solution of 12′-iodovinblastine (45 mg, 0.05 mmol) in dioxane (1mL) was added 3-methoxyphenylboronic acid (15 mg, 0.1 mmol) and Cs₂CO₃(78 mg, 0.24 mmol). The mixture was deoxygenated with an argon purge,and [1,1′-bis(diphenylphospino)ferrocene]dichloropalladium (5 mg, 0.006mmol) was added. The resulting mixture was deoxygenated again and thenheated to 60° C. for 4 h. The reaction mixture was cooled to roomtemperature, diluted with CH₂Cl₂, and filtered through diatomaceousearth. The filtrate was washed with water and brine, and then dried(MgSO₄). Purification by column chromatography (silica, 9:1 CH₂Cl₂/MeOH)followed by preparative TLC (silica, 7:3 EtOAc/MeOH) gave12′-(3-methoxyphenyl)vinblastine (18.4 mg, 41). This material wasdissolved in CH₂Cl₂ (1 mL) and treated with a drop of trifluoroaceticacid. The solution was evaporated to give12′-(3-methoxyphenyl)vinblastine trifluoroacetate (22 mg, quantitative):¹H NMR (300 MHz, CD₃OD) δ 9.55 (s, 1H), 7.61 (s, 1H), 7.32-7.19 (m, 3H),7.09 (d, J=8.0 Hz, 1H), 7.04 (dd, J=2.2, 1.8 Hz, 1H), 6.76 (d, J=7.5,1.8 Hz, 1H), 6.60 (s, 1H), 6.33 (s, 1H), 5.84 (dd, J=10.6, 4.3 Hz, 1H),5.57 (d, J=10.4 Hz, 1H), 5.27 (s, 1H), 4.61-4.54 (m, 1H), 3.91-3.56 (m,9H), 3.77 (s, 3H), 3.75 (s, 3H), 3.72 (s, 3H), 3.61 (s, 3H), 3.43-3.29(m, 2H), 3.15-3.09 (m, 2H), 2.80 (dd, J=14.4, 6.0 Hz, 1H), 2.68 (s, 3H),2.40 (dd, J=15.0, 4.7 Hz, 1H), 2.31-2.21 (m, 1H), 1.98-1.89 (m, 1H),1.98 (s, 3H), 1.71-1.62 (m, 1H), 1.58-1.56 (m, 2H), 1.51-1.39 (m, 3H),1.27-1.25 (m, 1H), 0.88 (t, J=7.4 Hz, 3H), 0.74 (t, J=7.2 Hz, 3H); ESIMS m/z 917 [M+H]⁺.

Example 10 Preparation of 12′-(4-Fluorophenyl)vinblastine

12′-(4-Fluorophenyl)vinblastine was prepared from 12′-bromovinblastineand 4-fluorophenyl boronic acid following the procedure described inExample 5 (63 mg, 43%). ¹H NMR (300 MHz, CDCl₃) δ 8.15 (s, 1H),7.60-7.52 (m, 3H), 7.41 (dd, J=8, 1 Hz, 1H), 722-7.08 (m, 3H), 6.99 (d,J=8 Hz, 2H), 6.85 (s, 1H), 5.91 (m, 1H), 5.56 (m, 1H), 5.19 (d, J=15 Hz,1H), 4.83 (m, 1H), 4.59 (d, J=15, 10 Hz, 1H), 4.10-3.60 (m, 3H), 3.93(s, 3H), 3.86 (s, 3H), 3.79 (s, 3H), 3.75 (s, 3H), 3.70 (s, 1H),3.60-3.00 (m, 8H), 3.11 (br s, 3H), 2.82 (m, 1H), 2.56 (m), 2.41 (m,1H), 2.12 (m, 1H), 2.07 (s, 3H), 1.96 (m, 1H), 1.67 (m, 2H), 1.55 (q,J=7 Hz, 2H), 1.47-1.19 (m, 2H), 0.99 (t, J=7 Hz, 3H) 0.76 (m, 3H); ESIm/z 905 [M+H]⁺.

Example 11 Preparation of 12′-(3-Fluorophenyl)vinblastineTrifluoroacetate

To a solution of 12′-iodovinblastine (45 mg, 0.05 mmol) in dioxane (1mL) was added 3-fluorophenylboronic acid (14 mg, 0.1 mmol) and Cs₂CO₃(80 mg, 0.25 mmol). The mixture was deoxygenated with an argon purge,and [1,1′-bis(diphenylphospino)ferrocene]dichloropalladium (5 mg, 0.006mmol) was added. The resulting mixture was deoxygenated again and thenheated to 60° C. for 4 h. The reaction mixture was cooled to roomtemperature, diluted with CH₂Cl₂ and filtered through diatomaceousearth. The filtrate was washed with water and brine, and then dried(MgSO₄). Purification by column chromatography (silica, 9:1 CH₂Cl₂/MeOH)followed by preparative TLC (silica gel, 7:3 EtOAc/MeOH) gave12′-(3-fluorophenyl)vinblastine (9.1 mg, 20%). This material wasdissolved in CH₂Cl₂ (1 mL) and treated with a drop of trifluoroaceticacid. The solution was evaporated to give12′-(3-fluorophenyl)vinblastine trifluoroacetate (11 mg, 80%): ¹H NMR(300 MHz, CD₃OD) δ 9.63 (s, 1H), 7.65 (s, 1H), 7.40-7.25 (m, 5H),6.94-6.88 (m, 1H), 6.60 (s, 1H), 6.33 (s, 1H), 5.84 (dd, J=10.6, 4.1 Hz,1H), 5.57 (d, J=10.3 Hz, 1H), 5.27 (s, 1H), 4.61-4.55 (m, 1H), 3.91-3.56(m, 9H), 3.77 (s, 3H), 3.72 (s, 3H), 3.61 (s, 3H), 3.43-3.30 (m, 2H),3.15-3.09 (m, 2H), 2.80 (dd, J=14.4, 6.3 Hz, 1H), 2.68 (s, 3H), 2.40(dd, J=16.3, 4.9 Hz, 1 H), 2.31-2.21 (m, 1H), 1.98-1.89 (m, 1H), 1.98(s, 3H), 1.71-1.62 (m, 1H), 1.58-1.56 (m, 2H), 1.50-1.39 (m, 3H),1.31-1.25 (m, 1H), 0.88 (t, J=7.3 Hz, 3H), 0.74 (t, J=7.1 Hz, 3H); ESIMS m/z 905 [M+H]⁺.

Example 12 Preparation of 12′-(3-Hydroxyphenyl)vinblastine

To a solution of 12′-iodovinblastine (39 mg, 0.040 mmol) in dioxane (1mL) was added 3-hydroxyphenylboronic acid (12 mg, 0.080 mmol) and Cs₂CO₃(68 mg, 0.21 mmol). The mixture was deoxygenated with argon, and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (4 mg,0.0040 mmol) was added. The resulting mixture was deoxygenated withargon again and then heated at 70° C. for 6 h and 80° C. overnight. Thereaction mixture was cooled to room temperature, diluted with CH₂Cl₂,and filtered through diatomaceous earth. The filtrate was washed withwater and brine, and dried. After removal of the solvents, the residuewas purified by column chromatography (silica, 9:1 CH₂Cl₂/MeOH) followedby preparative TLC (silica, 7:3 EtOAc/MeO) to give12-(3-hydroxyphenyl)vinblastine (4.2 mg, 12%): ¹H NMR (500 MHz, CD₃OD) δ7.51 (s, 1H), 7.24 (dd, J=14.0, 2.5 Hz, 1H), 7.14-7.09 (m, 2H),7.00-6.95 (m, 2H), 6.61 (dd, J=13.5, 7.5 Hz, 1H), 6.53 (s, 1H), 6.23 (s,1H), 5.75 (dd, J=17.0, 7.0 Hz, 1H), 5.28 (s, 1H), 5.19 (d, J=16.5 Hz,1H), 4.02-3.87 (m, 2H), 3.72-3.67 (m, 7H), 3.56-3.49 (m, 5H), 3.36-3.31(m, 1H), 3.20-2.96 (m, 4H), 2.73-2.61 (m, 7H), 2.40-2.31 (m, 2H),2.25-2.16 (m, 1H), 2.03-1.91 (m, 4H), 1.86-1.78 (m, 1H), 1.64-1.58 (m,1H), 1.44-1.19 (m, 7H), 0.83-0.69 (m, 6H); ESI MS m/z 903 [M+H]⁺.

Example 13 Preparation of 12′-(3-Pyridyl)vinblastine

12′-(3-Pyridyl)vinblastine was prepared from 12′-bromovinblastine and3-pyridyl boronic acid following the procedure described in Example 5(26 mg, 16%). ¹H NMR (300 MHz, CDCl₃) δ 9.82 (s, 1H), 8.89 (d, J=1.5 Hz,1H), 8.54 (dd, J=5, 1 Hz, 1H), 8.13 (s, 1H), 7.91 (dt, J=8, 2 Hz, 1H),7.69 (s, 1H), 7.33-7.39 (m, 2H), 7.22 (d, J=8 Hz, 1H), 6.63 (s, 1H),6.11 (s, 1H), 5.86 (dd, J=10, 4 Hz, 1H), 5.47 (s, 1H), 5.31 (d, J=10 Hz,1H), 3.99 (t, J=4 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.74 (s, 1H),3.68-3.87 (m, 2H), 3.63 (s, 3H), 3.26-3.43 (m, 5H), 3.18 (br s, 1H),3.14 (br s, 1H), 2.82 (br t, J=8 Hz, 1H), 2.71 (s, 3H), 2.68 (s, 1H),2.42-2.48 (m, 2H), 2.29 (br d, J=12 Hz, 1H), 2.15-2.22 (m, 1H), 2.11 (s,3H), 1.74-1.89 (m, 3H), 1.25-1.51 (m, 6H), 0.89 (t, J=7 Hz, 3H), 0.83(t, J=7 Hz, 3H). ESI m/z 888 [M+H]⁺.

Example 14 Preparation of 12′-(3-Thienyl)vinblastine

12′-(3-Thienyl)vinblastine was prepared from 12′-bromovinblastine and3-thienyl boronic acid by following the procedure described in Example 5and then converted to the free base by treatment with ammonium hydroxide(45 mg, 23%). ¹H NMR (300 MHz, CDCl₃) δ 9.87 (s, 1H), 8.07 (s, 1H), 7.68(s, 1H), 7.42 (m, 4H), 7.45 (d, J=7 Hz, 1H), 7.14 (d, J=8 Hz, 1H), 6.70(br s, 1H), 6.11 (s, 1H), 5.87 (dd, J=10, 4 Hz, 1H), 5.47 (s, 1H), 5.30(obs d, J=10 Hz, 1H), 5.30 (s, 1H), 4.00-3.85 (m, 1H), 3.80 (s, 6H),3.74 (s, 1H), 3.63 (s, 3H), 3.50-3.10 (m, 6H), 2.90-2.75 (m, 2H), 2.72(s, 3H), 2.68 (s, 1H), 2.53-2.37 (m, 2H), 2.36-2.00 (m, 2H), 2.11 (s,3H), 1.90-1.55 (m, 2H), 1.55-1.18 (m), 2.11 (s, 3H), 0.91 (t, J=7 Hz,3H), 0.82 (t, J=7 Hz, 3H); ESI MS m/z 893 [M+H]⁺.

Example 15 Preparation of 12′-(2-Thiazolyl)vinblastine

A solution of 12′-iodovinblastine (298 mg, 0.26 mmol) in THF (3 mL) wasdeoxygenated with an argon purge.Tetrakis(triphenylphosphine)palladium(0) (24 mg, 0.026 mmol) and2-thiazolylzinc bromide (1.68 mL, 1.06 mmol) was added and the mixtureheated at 60° C. overnight. The reaction mixture was diluted with brineand extracted with dichloromethane. The organic layers were combined andconcentrated under reduced pressure. Purification by reversed phasechromatography (C18, acetonitrile/water, 0.05% concentrated ammoniumhydroxide) gave 12′-(2-thiazolyl)vinblastine (32.8 mg, 14%). 1HNMR (300MHz, CD₃OD) 8.15 (s, 1H), 7.83 (d, J=5 Hz, 1H), 7.72 (dd, J=7, 2 Hz,1H), 7.53 (d, J=5 Hz, 1H), 7.36 (d, J=7 Hz, 1H), 6.51 (s, 1H), 6.38 (s,1H), 5.87 (dd, J=10, 3 Hz, 1H), 5.41 (d, J=10 Hz, 1H), 5.34 (s, 1H),4.66 (m, 1H), 4.23 (t, J=14 Hz, 1H), 3.85 (s, 3H), 3.78 (s, 3H), 3.70(s, 3H), 3.63 (s, 1H), 3.60-3.10 (m), 3.11-2.84 (m, 3H), 2.81 (s, 3H),2.61 (m, 1H), 2.51 (dd, J=15, 2 Hz, 1H), 2.17 (m, 1H), 2.15 (s, 1H),2.09 (s, 3H), 2.05 (m, 1H), 1.84 (m, 1H), 1.68 (m, 3H), 1.55-1.43 (m,4H), 1.01 (t, J=7 Hz, 3H), 0.74 (t, J=7 Hz, 3H); ESI m/z 894 [M+H]⁺.

Example 16 Preparation of 12′-(Trimethylsilylethynyl)vinblastine

A solution of 12′-iodovinblastine (310 mg, 0.33 mmol) in toluene (5 mL)and triethylamine (3 mL) was deoxygenated with an argon purge, copper(I) iodide (2.5 mg, 0.013 mmol) anddichlorobis(triphenylphosphine)palladium(II) (10 mg) were added and themixture deoxygenated again. Trimethylacetylene (0.06 mL, 0.42 mmol) wasadded and the mixture stirred at 55° C. for 24 h. After cooling, thereaction mixture was diluted with methanol and filtered throughdiatomaceous earth, concentrated to approximately 30 mL, filtered againand then concentrated to dryness. The mixture was diluted with 1 N HCland extracted with chloroform. The organic solution was separated andconcentrated under reduced pressure. Purification by flashchromatography (silica, 97:3 CHCl₃/MeOH) gave12′-(trimethylsilylethynyl)vinblastine (28 mg, quant). ¹H NMR (300 MHz,MeOD) δ 7.64 (s, 1H), 7.13 (s, 2H), 6.56 (s, 1H), 6.32 (s, 1H), 5.64(dd, J=10, 4 Hz, 1H), 5.35 (s, 1H), 5.29 (d, J=10 Hz, 1H), 4.02 (t, J=13Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.64 (s, 3H), 3.64 (m, 1H), 3.58(s, 1H), 3.42 (d, J=13 Hz, 1H), 3.23-3.12 (m), 3.05 (d, J=13 Hz, 1H),2.92-2.67 (m, 3H), 2.70 (s, 3H), 2.56-2.25 (m, 3H), 2.02 (s, 3H),1.92-1.25 (m, 6H), 0.90 (t, J=7 Hz, 3H), 0.75 (t, J=7 Hz, 3H), 0.22 (s,9H); ESI m/z 907 [M+H]⁺.

Example 17 Preparation of 12′-Ethynylvinblastine

To a solution of 12′-(trimethylsilylethynyl)vinblastine (0.088 g, 0.097mol) in methanol (1.8 mL) was added potassium carbonate (1.3 mg, 0.01mmol) and the mixture stirred at room temperature for 4 h. The mixturewas concentrated under reduced pressure, diluted with dichloromethane,washed with water and saturated sodium bicarbonate, dried (Na₂SO₄) andconcentrated under reduced pressure. Purification by reversed phasechromatography (C18, acetonitrile/water, 0.05% concentrated ammoniumhydroxide) gave 12′-(ethynyl)vinblastine 24.4 mg, 30%). ¹H NMR (300 MHz,MeOD) δ 7.60 (s, 1H), 7.16 (d, J=8 Hz, 1H), 7.13 (d, J=8 Hz, 1H), 6.58(s, 1H), 6.32 (s, 1H), 5.82 (dd, J=10, 4 Hz, 1H), 5.36 (s, 1H), 5.29 (d,J=10 Hz, 1H), 4.01 (t, J=13 Hz, 1H), 3.95 (m, 1H), 3.81 (s, 3H), 3.76(s, 3H), 3.66 (m, 1H), 3.65 (s, 3H), 3.58 (s, 1H), 3.45-3.12 (m), 3.02(d, J=13 Hz, 1H), 2.88-2.67 (m, 4H), 2.71 (s, 3H), 2.47 (m, 2H), 2.38(dd, J=13, 3 Hz, 1H), 2.03 (s, 3H), 1.87 (m, 1H), 1.66 (m, 1H),1.56-1.25 (m, 5H), 0.90 (t, J=7 Hz, 3H), 0.76 (t, J=7 Hz, 3H); ESI m/z835 [M+H]⁺.

Example 18 Preparation 12′-Propynylvinblastine Trifluoroacetate

Propynylmagnesium bromide (0.5 M in THF, 0.30 mL, 0.15 mmol) was addedto zinc bromide (0.5 M in THF, 0.30 mL, 0.15 mmol) in anhydrous1,4-dioxane (2 mL) under nitrogen. After 10 min[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (6.1 mg,0.008 mmol) was added followed by 12′-Iodovinblastine (71 mg, 0.076mmol) in anhydrous 1,4-dioxane (1 mL). The reaction mixture was heatedat 45° C. for 45 min then quenched by the addition of saturated aqueousNaHCO₃ (5 mL). After extraction with chloroform (3×8 mL) the combinedorganic extracts were washed with brine (5 mL), dried over MgSO₄, andevaporated to dryness in vacuo. The residue was purified by reversephase chromatography (C18, acetonitrile/water, 0.05% trifluoroaceticacid) to provide 12′-propynylvinblastine trifluoroacetate (38.6 mg, 47%)as a white powder after lyophilization: ¹H NMR (500 MHz, CD₃OD) δ 9.71(s, 1H), 7.56 (s, 1H), 7.20 (d, J=8.4 Hz, 1H), 7.12 (dd, J=8.4, 1.1 Hz,1H), 6.66 (s, 1H), 6.42 (s, 1H), 5.94 (dd, J=10.4, 4.4 Hz, 1H), 5.65 (d,J=10.7 Hz, 1H), 5.36 (s, 1H), 4.84 (m, 1H), 4.63 (dd, J=16.9, 11.5, 1H),3.96-3.56 (m, 7H), 3.86 (s, 3H), 3.82 (s, 3H), 3.69 (s, 3H), 3.48 (d,J=15.9 Hz, 1H), 3.19 (m, 3H), 2.88 (dd, J=14.4, 6.3 Hz, 1H), 2.77 (s,3H), 2.47 (dd, J=16.6, 4.7 Hz, 1H), 2.35 (m, 1H), 2.07 (s, 3H), 2.04 (m,2H), 2.01 (s, 3H), 1.74 (m, 1H), 1.66 (m, 2H), 1.52 (m, 3H), 1.38 (m,1H), 0.97 (t, J =7.4 Hz, 3H), 0.79 (t, J=7.3 Hz, 3H); ESI MS m/z 849[M+H]⁺.

Example 19 Preparation of 12′-(2-Phenylethynyl)vinblastine

12′-(Phenylethynyl)vinblastine was prepared from 12′-iodovinblastine andphenylacetylene following the procedure described in Example 16 exceptthat the reaction was run at room temperature (23 mg, 17%). ¹H NMR (300MHz, MeOD) δ 7.55 (s, 1H), 7.40 (dd, J=8, 2 Hz, 2H), 7.25 (m, 3H), 7.14(d, J=8 Hz, 1H), 7.08 (d, J=8 Hz, 1H), 6.50 (s, 1 H), 6.22 (s, 1H), 5.75(dd, J=10, 4 Hz, 1 H), 5.27 (s, 1H), 5.20 (d, J=10 Hz, 1H), 4.53 (br s,1H), 3.98 (t, J=14 Hz, 1H), 3.91 (m, 1H), 3.72 (s, 3H), 3.66 (s, 3H),3.55 (s, 3H), 3.49 (s, 1H), 3.32 (d, J=14 Hz, 1H), 3.25-3.05 (m, 5H),2.95 (d, J=14 Hz, 1H), 2.70 (m, 2H), 2.65 (s, 1H), 2.62 (s, 3H), 2.37(m, 2H), 2.07 (dm, J=14 Hz, 1H), 2.00 (m, 1H), 1.93 (s, 3H), 2.77 (m,1H), 1.57 (m, 1H), 1.50-1.15 (m, 5H), 0.78 (t, J=7 Hz, 3H), 0.68 (t, J=7Hz, 3H); ESI m/z 911 [M+H]⁺.

Example 20 Preparation of 12′-(3-Methylbutynyl)vinblastineTrifluoroacetate

12′-Iodovinblastine (52.7 mg, 0.056 mmol), copper(I) iodide (1.6 mg,0.0084 mmol), dichlorobis(triphenylphosphine)palladium(II) (3.9 mg,0.0056 mmol), toluene (1.2 mL), and triethylamine (0.8 mL) were combinedin a resealable glass test tube and argon was bubbled through thesolution for 10 min. 3-Methylbut-1-yne (22.9 mg, 0.33 mmol) was added,the test tube sealed, and the mixture was heated at 55° C. for 1.5 h.Saturated aqueous NaHCO₃ (5 mL) was added and the mixture was extractedwith ethyl acetate (3×5 mL). The combined organic extracts were washedwith brine (5 mL), dried over MgSO₄, and evaporated to dryness in vacuo.The residue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(3-methylbut-1-yne)vinblastine trifluoroacetate (24.0 mg, 40%) as awhite solid after lyophilization: ¹H NMR (300 MHz, CD₃OD) δ 7.56 (s,1H), 7.20 (d, J=8.5 Hz, 1H), 7.13-7.11 (m, 1H), 6.59 (s, 1H), 6.40 (s,1H), 5.91 (q, J=10.5, 6.0 Hz, 1H), 5.57-5.55 (m, 1H), 5.35 (s, 1H),4.84-4.81 (m, 1H), 4.67-4.59 (m, 1H), 3.97-3.80 (m, 9H), 3.68-3.58 (m,6H), 3.43-3.28 (m, 2H), 3.19-3.17 (m, 2H), 3.03-2.98 (m, 1H), 2.91-2.86(m, 1H), 2.78-2.74 (m, 4H), 2.50-2.47 (m, 1H), 2.33-2.28 (m, 1H),2.07-1.94 (m, 5H), 1.78-1.68 (m, 3H), 1.58-1.49 (m, 3H), 1.42-1.38 (m,1H), 1.24 (d, J=7.0 Hz, 6H), 0.97 (t, J=7.5 Hz, 3H), 0.77 (t, J=7.0 Hz,3H); ESI MS m/z 877 [M+H]⁺.

Example 21 Preparation of 12′-(3-Methylbutynyl)vincristine

A solution of 12′-iodovincristine (210 mg, 0.22 mmol) and triethylamine(3 mL) in THF (3 mL) was deoxygenated with argon for 20 min.Dichlorobis(triphenylphosphine)palladium(II) (9 mg, 0.01 mmol) andcopper(I) iodide was added, and the reaction mixture was again degassedwith argon for 10 min followed by addition of 3-methyl-1-butyne (0.05mL). The reaction mixture was heated at 60° C. overnight, cooled to roomtemperature, and quenched by the addition of 1 N HCl. The reactionmixture was extracted with methylene chloride and the organic layer wasdried (sodium sulfate), filtered, and concentrated under reducedpressure to give a light orange solid. Purification by flash columnchromatography (silica, 97:3 to 95:% CH₂Cl₂/MeOH) gave12′-(3-methyl-butynl)vincristine (22 mg, 11%) as an orange solid: ¹H NMR(300 MHz, CD₃OD) δ 8.99 (s, 1H), 7.45 (s, 1H), 7.21-7.15 (m, 2H), 7.06(d, J=7 Hz, 1H), 6.90 (s, 1H), 5.90 (m, 1H), 5.40 (d, J=6 Hz, 1H),4.13-4.03 (m, 3H), 3.89 (s, 3H), 3.88 (m, 1H), 3.74 (m, 1H), 3.71 (s,3H), 3.62 (s, 3H), 3.40 (m), 3.01 (m, 3H), 2.90-2.74 (m, 5H), 2.63 (m,1H), 2.41 (m, 1H), 2.27 (m, 1H), 2.07 (m, 5H), 1.78 (m, 1H), 1.51-1.30(m), 1.28 (d, J=7 Hz, 6H), 0.89 (t, J=7 Hz, 3H), 0.76 (t, J=7 Hz, 3H);ESI MS m/z 891 [M+]⁺.

Example 22 Preparation of 12′-(Hexynyl)vincristine

12′-(Hexynyl)vincristine was prepared from 12′-iodovinblastine followingthe procedure described in Example 16 (12 mg, 44%). ¹H NMR (300 MHz,CD₃OD) δ 8.95 (s, 1H), 7.54 (s, 1H), 7.24 (s, 1H), 7.23 (d, J=8 Hz, 1H),7.11 (d, J=8 Hz, 1H), 6.86 (s, 1H), 5.88 (d, J=10, 5 Hz, 1H), 5.41 (d,J=10 Hz, 1H), 5.14 (s, 1H), 4.57 (s, 1H), 4.40 (dd, J=16, 11 Hz, 1H),3.98 (t, J=15 Hz, 1H), 3.92 (s, 3H), 3.88 (s, 1H), 3.70 (s, 3H), 3.62(s, 3H), 3.52 (m, 2H), 3.40-2.84 (m, 5H), 2.77 (m, 1H), 2.40 (m, 1H),2.40 (t, J=7 Hz, 2H), 2.12 (m, 1H), 2.01 (m, 1H), 2.00 (s, 3H), 1.83 (m,1H), 1.66-1.20 (m, 14H), 0.96 (t, J=7 Hz, 3H), 0.93 (t, J=7 Hz, 3H),0.76 (t, J=7 Hz, 3H); ESI MS m/z 905 [M+H]⁺.

Example 23 Preparation of 12′-(Hexynyl)vinblastine

12′-(Hexynyl)vinblastine was prepared from 12′-iodovinblastine followingthe procedure described in Example 16 (19 mg, 14%). ¹H NMR (300 MHz,CD₃OD) δ 7.77 (s, 1H), 7.26 (d, J=8 Hz, 1H), 7.14 (d, J=8 Hz, 1H), 6.32(s, 1H), 6.25 (s, 1H), 5.83 (d, J=10, 5 Hz, 1H), 5.77 (d, J=5 Hz, 1H),5.30 (s, 1H), 5.23 (d, J=10 Hz, 1H), 4.41 (s, 1H), 3.85 (s, 3H), 3.77(m, 1H), 3.75 (s, 3H), 3.73 (s, 3H), 3.58 (s, 1H), 3.41 (m, 2H), 3.20(m, 1H), 3.06 (m, 1H), 2.71 (s, 3H), 2.70-2.44 (m, 3H), 2.41 (t, J=7 Hz,2H), 2.26 (m, 1H), 2.05 (m, 3H), 2.01 (s, 3H), 1.90 (br s, 1H), 1.79 (m,1H), 1.66-1.20 (m, 10H), 0.96 (t, J=7 Hz, 3H), 0.93 (t, J=7 Hz, 3H),0.76 (t, J=7 Hz, 3H); ESI MS m/z 859 [M+H]⁺.

Example 24 Preparation of 12′-(N,N-Dimethylaminopropynyl)vinblastineTrifluoroacetate

12′-iodovinblastine (52.8 mg, 0.057 mmol), copper(I) iodide (1.6 mg,0.0086 mmol), dichlorobis(triphenylphosphine)palladium(II) (4.0 mg,0.0057 mmol), toluene (1.2 mL), and triethylamine (0.8 mL) were combinedin a resealable glass test tube and argon was bubbled through thesolution for 10 min. 1-Dimethylamino-2-propyne (36.7 μL, 0.33 mmol) wasadded, the test tube sealed, and the mixture was heated at 55° C. for1.5 h. Saturated aqueous NaHCO₃ (5 mL) was added, and the mixture wasextracted with ethyl acetate (3×5 mL). The combined organic extractswere washed with brine (5 mL), dried over MgSO₄, and evaporated todryness in vacuo. The residue was purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid) toprovide 12′-(N,N-dimethylaminopropynyl)vinblastine trifluoroacetate(22.4 mg, 33%), which was a white solid after lyophilization: ¹H NMR(300 MHz, CD₃OD) δ 9.91 (br s, 1H), 7.76 (s, 1H), 7.30-7.26 (m, 2H),6.58 (s, 1H), 6.38 (s, 1H), 5.90 (t, J=10.5, 6.0 Hz, 1H), 5.52 (d, J=9.0Hz, 1H), 5.33 (s, 1H), 4.90-4.81 (m, 1H), 4.66-4.61 (m, 1H), 4.27 (s,2H), 3.96-3.84 (m, 5H), 3.79 (s, 3H), 3.69-3.62 (m, 7H), 3.55-3.48 (m,1H), 3.33-2.29 (m, 1H), 3.20-3.16 (m, 3H), 3.00 (s, 6H), 2.89-2.85 (m,1H), 2.75 (s, 3H), 2.50-2.45 (m, 1H), 2.24-2.21 (m, 1H), 2.05 (s, 3H),2.03 (s, 1H), 1.97-1.89 (m, 1H), 1.74-1.67 (m, 3H), 1.57-1.49 (m, 3H),1.40-1.45 (m, 1H), 0.96 (t, J=7.5 Hz, 3H), 0.75 (t, J=7.5 Hz, 3H); ESIMS m/z 892 [M+H]⁺.

Example 25 Preparation of 12′-vinylvinblastine

A solution of 12′-iodovinblastine (39 mg, 0.042 mmol) in DME (0.5 mL)and water (0.2 mL) was deoxygenated with argon for 3 minutes. Thereaction vessel was charged with 2,4,6-trivinylcyclotriboroxane pyridinecomplex (11 mg, 0.46 mmol), tetrakis(triphenylphosphine)palladium(0)(6.3 mg, 0.050 mmol), K₂CO₃ (6.4 mg, 0.046 mmol) and the mixture washeated to 80-90° C. After 2 h, the reaction appeared complete by ESImass spectral analysis. The reaction mixture was diluted with saturatedaqueous NaHCO₃ (8 mL) and extracted with EtOAc (2×2 mL). The combinedextracts were dried (Na₂SO₄) and concentrated to a brown solid which waspurified by flash chromatography (silica, [CHCl₃/MeOH/NH₄OH(40:18:2)]/CH₂Cl₂, 1:99 to 10:90) to yield 12′-vinylvinblastine (14 mg,31%) as a white solid: ¹H NMR (500 MHz, CD₃OD) δ 9.63 (br s, 1H), 7.51(s, 1H), 7.33-7.31 (m, 1H), 7.23 (d, J=8.5 Hz, 1H), 6.81 (dd, J=17.5,4.4 Hz, 1H), 6.67 (s, 1H), 6.42 (s, 1H), 5.97-5.92 (m, 1H), 5.70-5.64(m, 2H), 5.35 (s, 1H), 5.08 (d, J=11.5 Hz, 1H), 4.67-4.60 (m, 1H),3.97-3.88 (m, 2H), 2.87-2.81 (m, 6H), 3.75-3.58 (m, 6H), 3.50-3.47 (m,1H), 3.37-3.33 (m, 1H), 3.23-3.17 (m, 3H), 2.91-2.88 (m, 1H), 2.78-2.77(m, 4H), 2.49-2.44 (m, 1H), 2.37-2.32 (m, 1H), 2.08-2.00 (m, 5H),1.78-1.67 (m, 3H), 1.57-1.36 (m, 5H), 0.97 (t, J=7.5 Hz, 3H), 0.81 (t,J=7.5 Hz, 3H); ESI MS m/z 837 [M+H]⁺.

Example 26 Preparation of 12′-(2-Ethoxycarbonylvinyl)vinblastine

A solution of 12′-iodovinblastine (0.248 g, 0.26 mmol) in toluene (2 mL)was deoxygenated with argon. Palladium acetate (3 mg, 0.013 mmol),triphenylphosphine (32 mg, 0.12 mmol), and triethylamine (0.05 mL, 0.36mmol) were added and the mixture deoxygenated again. After heating to70° C., ethyl acrylate (0.058 mL, 0.53 mmol) was added and the mixturestirred overnight. The mixture was then cooled to room temperature,diluted with dichloromethane, filtered through diatomaceous earth, andconcentrated under reduced pressure. Ethyl acetate was added and themixture filtered through diatomaceous earth and concentrated underreduced pressure. Purification by reversed phase chromatography (C18,acetonitrile/water, 0.05% concentrated ammonium hydroxide) gave12′-(2-ethoxycarbonylvinyl)vinblastine (24.3 mg, quant.). ¹H NMR (300MHz, MeOD) δ 7.80 (d, J=16 Hz, 1H), 7.62 (s, 1H), 7.41 (d, J=8 Hz, 1H),7.21 (d, J=8 Hz, 1H), 6.58 (s, 1H), 6.39 (d, J=16 Hz, 1H), 6.32 (s, 1H),5.80 (dd, J=10, 4 Hz, 1H), 5.36 (s, 1H), 5.30 (d, J=10 Hz, 1H), 4.23 (q,J=7 Hz, 2H), 4.05 (t, J=13 Hz, 1H), 3.95 (m, 1H), 3.81 (s, 3H), 3.76 (s,3H), 3.66 (m, 1H), 3.65 (s, 3H), 3.58 (s, 1H), 3.02 (d, J=14 Hz, 1H),3.34-3.15 (m, 4H), 3.07 (br d, J=13 Hz, 1H), 2.88-2.65 (m, 4H), 2.72 (s,3H), 2.46 (m, 2H), 2.38 (dd, J=16, 5 Hz, 1H), 2.08 (m, 2H), 2.03 (s,3H), 1.87 (m, 1H), 1.66 (m, 1H), 1.56-1.25 (m, 5H), 1.32 (t, J=7 Hz,3H), 0.90 (t, J=7 Hz, 3H), 0.77 (t, J=7 Hz, 3H); ESI m/z 909 [M+H]⁺.

Example 27 Preparation of 12′-(2-tert-Butoxycarbonylvinyl)vinblastine

To a solution of 12′-iodovinblastine (114 mg, 0.122 mmol) in toluene (2mL) was added palladium acetate (2 mg, 0.009 mmol), triphenylphosphine(3.1 mg, 0.005 mmol), triethylamine (24 μL, 0.172 mmol) and the reactionmixture was deoxygenated with an argon purge. Tert-butyl acrylate (36μL, 0.246 mmol) was added and the reaction mixture was heated to 70° C.After 1 h, HPLC analysis indicated that 12′-iodovinblastine was presentso another equivalent of all reagents except 12′-iodovinblastine wereadded and the reaction mixture was heated to 70° C. for an additional 17h. The reaction was cooled to room temperature, the mixture diluted withCH₂Cl₂ (15 mL) and then filtered through diatomaceous earth. Thefiltrate was concentrated to dryness, and the resulting residue wastriturated with 9:1 ethyl acetate/methanol and the solid was removed byfiltration. The filtrate was concentrated and the residue purified bycolumn chromatography (silica, 9:1 EtOAc/MeOH) to give an orange solidwhich was further purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% concentrated ammonium hydroxide) to give12′-(2-tert-butoxycarbonylviny)vinblastine as a tan solid (13 mg, 11%):¹H-NMR (300 MHz, CD₃OD) δ 7.62 (d, J=15.9 Hz, 1H), 7.57 (s, 1H), 7.31(d, J=8.5 Hz, 1H), 7.13 (d, J=8.8 Hz, 1H), 6.51 (s, 1H), 6.26 (d, J=15.8Hz, 1H), 6.25 (s, 1H), 5.77 (dd, J=10.2, 4.2 Hz, 1H), 5.29 (s, 1H), 5.23(d, J=10.2 Hz, 1H), 4.02-3.86 (m, 2H), 3.74 (s, 3H), 3.69 (s, 3H), 3.57(s, 3H), 3.51 (s, 1H), 3.34-3.13 (m, 3H), 3.02-2.96 (m, 1H), 2.79-2.64(m, 3H), 2.64 (s, 3H), 2.40-2.31 (m, 2H), 2.24-2.19 (m, 1H), 2.06-1.95(m, 2H), 1.95 (s, 3H), 1.84-1.74 (m, 1H), 1.67-1.55 (m, 1H), 1.46 (s,9H), 1.46-1.21 (m, 8H), 0.83 (t, J=5.0 Hz, 3H), 0.70 (t, J=6.9 Hz, 3H);ESI MS m/z 938 [M₁+H]⁺.

Example 28 Preparation of 12′-(2-Carboxyvinyl)vinblastine

A mixture of 12′-(2-tert-butoxycarbonylvinyl)vinblastine (17 mg, 0.018mmol) in CH₂Cl₂ (1 mL) was treated with trifluoroacetic acid (45 μL,0.069 mmol) at room temperature. After 3.5 h, HPLC indicated that thereaction was complete. The reaction was quenched with saturated aqueoussodium bicarbonate, diluted with CH₂Cl₂ and the layers were separated.The aqueous phase was extracted with CH₂Cl₂ (2×10 mL). The combinedorganics were dried over MgSO₄, filtered and concentrated to give12′-(2-carboxyvinyl)vinblastine as a brown solid (9.4 mg, 59%): ¹H NMR(300 MHz, CD₃OD) δ 7.49 (s, 1H), 7.44 (d, J=15.9 Hz, 1H), 7.26 (d, J=8.5Hz, 1H), 7.11-7.02 (m, 2H), 6.50 (s, 1H), 6.34 (d, J=15.8 Hz, 1H), 6.22(s, 1H), 5.75 (dd, J=10.2, 4.6 Hz, 1H), 5.27 (s, 1H), 5.20 (d, J=9.9 Hz,1H), 4.75-4.65 (m, 1H), 4.00-3.85 (m, 2H), 3.71 (s, 3H), 3.65 (s, 3H),3.55 (s, 3H), 3.47 (s, 2H), 3.33 (d, J=14.3 Hz, 1H), 2.99 (d, J=13.3 Hz,1H), 2.61 (s, 3H), 2.77-2.52 (m, 6H), 2.39-2.34 (m, 2H), 2.23-2.17 (m,2H), 1.93 (s, 3H), 1.84-1.74 (m, 1H), 1.62-1.53 (m, 1H), 1.47-1.18 (m,6H), 0.81 (t, J=7.1 Hz, 3H), 0.69 (t, J=7.0 Hz, 3H); ESI MS m/z 881[M+H]⁺.

Example 29 Preparation of 12′-(3-Oxohex-1-enyl)vinblastine

12′-(3-Oxohex-1-enyl)vinblastine was prepared from 12′-iodovinblastineand hexen-3-one following the procedure described in Example 26, yield(90 mg, 42%). ¹H NMR (300 MHz, MeOD) δ 7.80 (d, J=16 Hz, 1H), 7.73 (s,1H), 7.45 (d, J=8 Hz, 1H), 7.22 (d, J=8 Hz, 1H), 6.39 (d, J=16 Hz, 1H),6.58 (s, 1H), 6.32 (s, 1H), 5.84 (dd, J=10, 4 Hz, 1H), 5.49 (s, 1H),5.34 (d, J=10 Hz, 1H), 4.06 (t, J=13 Hz, 1H), 3.95 (m, 1H), 3.81 (s,3H), 3.76 (s, 3H), 3.65 (s, 3H), 3.64 (m, 1H), 3.58 (s, 1H), 3.39 (d,J=14 Hz, 1H), 3.36-3.15 (m, 4H), 3.07 (br d, J=14 Hz, 1H), 2.88-2.54 (m,3H), 2.70 (s, 3H), 2.69 (t, J=7 Hz, 2H), 2.42 (m, 2H), 2.28 (dd, J=16, 5Hz, 1H), 2.08 (m, 2H), 2.03 (s, 3H), 1.87 (m, 1H), 1.68 (sex, J=7 Hz,2H), 1.66 (m, 1H), 1.56-1.25 (m, 6H), 0.99 (t, J=7 Hz, 3H), 0.88 (t, J=7Hz, 3H), 0.77 (t, J=7 Hz, 3H); ESI m/z 907 [M+H]⁺.

Example 30 Preparation of 12′-(2-Cyanovinyl)vinblastine

12′-(2-cyanovinyl)vinblastine was prepared from 12′-iodovinblastine andacrylonitrile following the procedure described in Example 26, yield (10mg, 4%): ¹H NMR (300 MHz, MeOD) δ 7.67 (s, 1H), 7.62 (d, J=17 Hz, 1H),7.37 (d, J=8 Hz, 1H), 7.24 (d, J=8 Hz, 1H), 6.57 (s, 1H), 6.31 (s, 1H),6.06 (d, J=17 Hz, 1H), 6.29 (m, 1H), 5.84 (dd, J=10, 4 Hz, 1H), 5.35 (s,1H), 5.31 (d, J=10 Hz, 1H), 4.00 (m, 2H), 3.81 (s, 3H), 3.75 (s, 3H),3.64 (s, 3H), 3.64 (m, 1H), 3.58 (s, 1H), 3.38 (d, J=14 Hz, 1H),3.36-3.15 (m, 2H), 3.07 (br d, J=14 Hz, 1H), 2.88-2.54 (m, 2H), 2.73 (s,1H), 2.72 (s, 3H), 2.45 (m, 2H), 2.27 (br d, J=14 Hz, 1H), 2.08 (m, 2H),2.03 (s, 3H), 1.84 (m, 1H), 1.66 (m, 1H), 1.56-1.25 (m, 6H), 0.90 (t,J=7 Hz, 3H), 0.75 (t, J=7 Hz, 3H); ESI m/z 862 [M+H]⁺.

Example 31 Preparation of 12′-(3-tert-Butoxycarbonylaminopropenyl)vinblastine

12′-(3-tert-Butoxycarbonylaminopropenyl)vinblastine was prepared from12′-iodovinblastine and t-butyl-N-allylcarbonate following the proceduredescribed in Example 26, yield (24 mg, 9%): ¹H NMR (300 MHz, MeOD) δ7.41 (s, 1H), 7.20 (d, J=8 Hz, 1H), 7.08 (d, J=8 Hz, 1H), 6.60 (d, J=15Hz, 1H), 6.60 (s, 1H), 6.33 (s, 1H), 6.10 (dt, J=15, 6 Hz, 1H), 5.84(dd, J=10, 4 Hz, 1H), 5.49 (s, 1H), 5.36 (s, 1H), 5.28 (d, J=10 Hz, 1H),4.22 (d, J=5 Hz, 2H), 4.04 (m, 2H), 3.81 (s, 3H), 3.81 (m, 1H), 3.76 (s,3H), 3.64 (s, 3H), 3.58 (s, 1H), 3.42 (d, J=15 Hz, 1H), 3.36-3.15 (m,4H), 3.04 (br d, J=15 Hz, 1H), 2.78 (m, 2H), 2.73 (s, 1H), 2.71 (s, 3H),2.44 (m, 2H), 2.28 (br d, J=14 Hz, 1H), 2.08 (m, 1H), 2.03 (s, 3H), 1.87(m, 1H), 1.66 (m, 1H), 1.56-1.25 (m, 6H), 1.48 (s, 9H), 0.90 (t, J=7 Hz,3H), 0.78 (t, J=7 Hz, 3H); ESI m/z 966 [M+H]⁺.

Example 32 Preparation of 12′-(4-Hydroxybutylsulfanyl)vinblastine

A flask containing compound 12′-iodovinblastine (200 mg, 0.214 mmol),tris(dibenzylideneacetone)dipalladium(0) (19.6 mg, 0.0214 mmol) and1,1′-bis(diphenylphosphino)ferrocene (47.5 mg, 0.0856 mmol) was flushedwith argon. Triethylamine (47 μL, 0.428 mmol), N-methylpyrrolidine (4mL) and 4-mercaptobutan-1-ol (44 μL, 0.428 mmol) were added by syringeand the mixture was heated at 60° C. for 2 d. After cooling, the mixturewas diluted with dichloromethane and washed with brine. The aqueouslayer was extracted with dichloromethane and the combined organic layersconcentrated under reduced pressure and dried under high vacuum.Purification by reversed phasechromatography (C18, acetonitrile/water,0.05% concentrated ammonium hydroxide) gave12′-(4-hydroxybutylsufanyl)vinblastine (0.030 g, 15%). ¹H NMR (300 MHz,CDCl₃) δ 9.83 (s, 1H), 8.04 (s, 1H), 7.60 (s, 1H), 7.23 (dd, J=8, 1 Hz,1H), 7.04 (d, J=7 Hz, 1H), 6.58 (s, 1H), 6.09 (s, 1H), 5.87 (dd, J=10, 4Hz, 1H), 5.46 (s, 1H), 5.29 (d, J=10 Hz, 1H), 3.97 (t, J=15 Hz, 1H),3.80-3.60 (m, 3H), 3.79 (s, 6H), 3.73 (s, 1H), 3.62 (s, 3H), 3.38-3.29(m, 4H), 3.09 (m, 2H), 2.93 (m, 2H), 2.85 (s, 1H), 2.84 (s, 3H), 2.80(br s, 2H), 2.65 (s, 1H), 2.43 (m, 2H), 2.25 (d, J=15 Hz, 1H), 2.21 (m,1H), 2.11 (s, 3H), 1.90-1.20 (m), 1.48-1.28 (m, 6H), 0.89 (t, J=7 Hz,3H), 0.76 (t, J=7 Hz, 3H); ESI MS m/z 915 [M+H]⁺.

Example 33 Preparation of 12′-(4-Hydroxypropylsulfanyl)vinblastine

12′-(4-Hydroxypropylsufanyl)vinblastine was prepared from12′-iodovinblastine and 3-mercaptopropan-1-ol following the proceduredescribed in Example 33, yield (16 mg, 8%): ¹H NMR (300 MHz, CDCl₃) δ9.68 (s, 1H), 8.74 (s, 1H), 7.42 (s, 1H), 7.24 (d, J=8 Hz, 1H), 7.06(dd, J=8, 1 Hz, 1H), 6.54 (s, 1H), 6.36 (s, 1H), 5.78 (dd, J=10, 4 Hz,1H), 5.29 (d, J=10 Hz, 1H), 5.14 (s, 1H), 4.48 (t, J=5 Hz, 1H), 4.01 (m,2H), 3.74 (s, 3H), 3.67 (s, 3H), 3.55 (s, 3H), 3.53 (s, 1H), 3.45 (q,J=6 Hz, 1H), 3.40-3.30 (m, 5H), 2.95-2.60 (m, 7H), 2.87 (t, J=7 Hz, 2H),2.65 (s, 3H), 2.55-2.30 (m, 5H), 2.02 (m, 1H), 2.01 (s, 3H), 1.64 (m,2H), 1.53 (m, 1H), 1.31 (m, 1H), 1.19 (m, 2H), 0.78 (t, J=7 Hz, 3H),0.66 (t, J=7 Hz, 3H); ESI MS m/z 901 [M+H]⁺.

Example 34 Preparation of12′-(3-Methanesulfonyloxypropylsulfanyl)vinblastine

A mixture of 12′-(3-hydroxypropylthio)vinblastine (77 mg, 0.085 mmol)and triethylamine (24 μL, 0.17 mmol) in dichloromethane (2.0 mL) wastreated with methanesulfonyl chloride (7.6 μL, 0.097 mmol) at 0° C. andthe reaction was stirred at room temperature for 4 h. The reactionmixture was diluted with dichloromethane and the mixture was washed withsaturated NaHCO₃ and brine. The organic layer was dried over MgSO₄ andthen concentrated to afford an orange solid. Purification by columnchromatography (silica gel, CH₂Cl₂/CH₃OH, 39:1) gave12′-(3-methanesulfonyloxypropylsulfanyl)vinblastine as an off-whitesolid (40 mg, 48%): ¹H NMR (300 MHz, CD₃OD) δ 7.59 (s, 1H), 7.22 (dd,J=8.4, 1.4 Hz, 1H), 7.16 (d, J=8.3 Hz, 1H), 6.62 (s, 1H), 6.33 (s, 1H),5.86 (dd, J=9.9, 3.7 Hz, 1H), 5.38 (s, 1H), 5.31, (d, J=10.2 Hz, 1H),4.35 (t, J=6.1 Hz, 2H), 4.15-4.06 (m, 1H), 4.01-3.91 (m, 1H), 3.82 (s,3H), 3.78 (s, 3H), 3.65 (s, 3H), 3.70-3.60 (m, 1H), 3.43 (m, 10H),3.08-2.93 (m, 2H), 3.02 (s, 3H), 2.87-2.70 (m, 2H), 2.73 (s, 3H),2.49-2.54 (m, 1H), 2.32-2.25 (m, 1H), 2.15-1.84 (m, 3H), 2.04 (s, 3H),1.73-1.64 (m, 1H), 1.54-1.21 (m, 6H), 0.91 (t, J=7.3 Hz, 3H), 0.79 (t,J=7.2 Hz, 3H); ESI MS m/z 979 [M+H]⁺.

Example 35 Preparation of 12′-(2-Hydroxyethylsulfanyl)vinblastine

12′-(2-Hydroxyethylsulfanyl)vinblastine was prepared from12′-iodovinblastine and 2-mercaptoethanol following the proceduredescribed in Example 33, yield (33 mg, 17%) as an off-white powder: ¹HNMR (300 MHz, DMSO-d₆) δ 10.05 (br s, 1H), 8.97 (br s, 1H), 7.64 (s,1H), 7.31 (d, J=8 Hz, 1H), 7.18 (dd, J=8, 1 Hz, 1H), 6.40 (s, 1H), 5.81(dd, J=10, 4 Hz, 1H), 5.34 (br s, 1H), 5.01 (m, 2H), 4.82 (m, 1H), 4.34(m, 1H), 3.85 (t, J=15 Hz, 1H), 3.80-3.10 (m), 3.77 (s, 3H), 3.66 (s,3H), 3.60 (s, 3H), 3.02-2.98 (m, 4H), 2.71 (m, 3H), 2.15 (m, 2H), 2.01(s, 3H), 1.73 (m, 1H), 1.60-1.35 (m, 6H), 1.16 (m, 1H), 0.88 (t, J=8 Hz,3H), 0.67 (t, J=8 Hz, 3H); ESI MS m/z 887 [M+H]⁺.

Example 36 Preparation of 12′-(4-Methoxybenzylsulfanyl)vinblastine

A mixture of 12′-iodovinblastine (200 mg, 0.214 mmol),1,1′-bis(diphenylphosphino)ferrocene (48 mg, 0.09 mmol), andtris(dibenzylideneacetone)dipalladium(0) (20 mg, 0.02 mmol) in NMP (4mL) was deoxygenated with argon for 10 min. Triethylamine (47 μL) andthiol (66 mg, 0.428 mmol) were added and the reaction mixture was heatedto 60° C. for 72 h. The reaction mixture was cooled to room temperature,and partitioned between brine and methylene chloride. The organic layerwas separated and concentrated under reduced pressure. The resultingresidue was purified by chromatography (silica, 9:1 CH₂Cl₂/MeOH), thenfurther purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% concentrated ammonium hydroxide) to provide12′-(4-methoxybenzylsulfanyl)vinblastine (3 mg, 1%) as a white solid: ¹HNMR (300 MHz, CDCl₃) δ 8.08 (br s, 1H), 7.45 (s, 1H), 7.22 (d, J=9 Hz,2H), 7.03 (d, J=9 Hz, 1H), 6.85 (d, J=8 Hz, 2H), 6.39 (s, 1H), 6.09 (s,1H), 5.90 (dd, J=10, 4 Hz, 1H), 5.39 (m, 2H), 4.46 (m, 1H), 4.11 (s,2H), 3.89-3.35 (m), 3.68 (s, 3H), 3.28 (m, 2H), 3.09-2.84 (m, 5H), 2.47(d, J=16 Hz, 1H), 2.27 (m, 1H), 2.13 (s, 3H), 2.00-1.20 (m), 0.99 (t,J=8 Hz, 3H), 0.73 (t, J=7 Hz, 3H); ESI MS m/z 963 [M+H]⁺.

Example 37 Preparation of 12′-(2-Chlorobenzylsulfanyl)vincristine

A solution of 11′-iodovincristine (60 mg, 0.063 mmol) in NMP (3 mL) wasdeoxygenated with argon for 10 minutes. The reaction vessel was chargedwith 1,1′-bis(diphenylphosphino)ferrocene (20 mg, 0.035 mmol),tris(dibenzylideneacetone)dipalladium(0) (9 mg, 0.009 mmol) and Et₃N (13mg, 0.13 mmol). The mixture was stirred for 10 min at room temperature;2-chlorobenzenmethane-thiol (20 mg, 0.126 mmol) was added and then themixture stirred at 60° C. overnight. The mixture was cooled to roomtemperature, diluted with methylene chloride (100 mL) and washed withsaturated aqueous NH₄Cl (3×10 mL) and brine (3×10 mL). The organic layerwas dried (Na₂SO₄), filtered and concentrated. The residue was purifiedby flash chromatography (silical, 10:1 CH₂Cl₂/MeOH) and then reversephase chromatography (C-18, acetonitrile/water, 0.05% trifluoroaceticacid) to give 12 mg of white solid. This solid was dissolved in 10 mL ofEtOAc and stirred with 50 mg of solid NaHCO₃ for 1 h. The mixture wasfiltered, concentrated to give 12′-(2-chlorobenzylsulfanyl)vincristine(9.7 mg, 15%) as an off-white solid: mp 168-170° C.; ¹H NMR (500 MHz,CD₃OD) δ 8.95 (s, 1H), 7.46 (s, 1H), 7.37 (d, J=8.0 Hz, 1H), 7.36 (s,1H), 7.25 (d, J=8.5 Hz, 1H), 7.20-7.11 (m, 4H), 6.98 (s, 1H), 5.97 (dd,J=11.0, 5.0 Hz, 1H), 5.74 (d, J=10.5 Hz, 1H), 5.48 (s, 1H), 5.18 (s,1H), 4.67 (s, 1H), 4.64 (dd, J=17.0, 11.0 Hz, 1H), 4.15 (q, J=11.0 Hz,2H), 4.08 (s, 1H), 3.99-3.94 (m, 2H), 3.92 (s, 3H), 3.68-3.81 (m, 3H),3.73 (s, 3H), 3.68 (s, 3H), 3.57 (dd, J=11.2, 6.5 Hz, 1H), 3.54-3.49 (m,2H), 3.34 (dd, J=7.5, 5.0 Hz, 1H), 3.22 (dd, J=17.5, 6.5, 1H), 3.16 (s,1H), 2.84 (dd, J=14.5, 3.6 Hz, 1H), 2.51-2.47 (m, 1H), 2.42-2.34 (m,1H), 2.02 (s, 3H), 1.99-1.92 (m, 1H), 1.67-1.60 (m, 3H), 1.51 (q, J=7.5Hz, 3H), 1.32-1.28 (m, 1H), 0.96 (t, J=7.0 Hz, 3H), 0.80 (t, J=7.5 Hz,3H); ESI MS m/z 981 [M+H]⁺.

Example 38 Preparation of 12′-(2-Fluorobenzylsulfanyl)vincristineTrifluoroacetate

A solution of 12′-iodovincristine (50 mg, 0.053 mmol) in NMP (1.5 mL)was deoxygenated with argon for 10 minutes. The reaction vessel wascharged with 1,1′-bis(diphenylphosphino)ferrocene (20 mg, 0.035 mmol),tris(dibenzylideneacetone)dipalladium(0) (10 mg, 0.011 mmol) and Et₃N(13 mg, 0.13 mmol). The mixture was stirred for 20 min at roomtemperature, 2-fluorophenylmethane-thiol (20 mg, 0.14 mmol) was addedand then the mixture stirred at 60° C. overnight. The mixture was cooledto room temperature, diluted with EtOAc (100 mL) and washed withsaturated aqueous NH₄Cl (3×10 mL) and brine (3×10 mL). The organic layerwas dried (Na₂SO₄), filtered and concentrated. The residue was purifiedby chromatography (silica, 4:1 CH₂Cl₂/MeOH), then further purified byreverse phase chromatography (C-18, acetonitrile/water, 0.05%trifluoroacetic acid) to give 12′-(2-fluorobenzylsulfanyl)vincristine (8mg, 15%) as a light brown solid: ¹H NMR (500 MHz, CD₃OD) δ 9.98 (s, 1H),8.99 (s, 1H), 7.46 (s, 1H), 7.34 (s, 1H), 7.28-7.13 (m, 4H), 7.04-6.99(m, 3H), 5.98 (dd, J=10.5, 5.5 Hz, 1H), 5.73 (d, J=10.0 Hz, 1H), 5.18(s, 1H), 4.67 (s, 1H), 4.64 (dd, J=17.5, 11.0 Hz, 1H), 4.09-4.04 (m,3H), 3.99-3.94 (m, 5H), 3.86-3.79 (m, 2H), 3.76-3.73 (m, 1H), 3.72 (s,3H), 3.67 (s, 3H), 3.58-3.50 (m, 3H), 3.40-3.37 (m, 1H), 3.23 (dd,J=7.5, 5.5 Hz, 1H), 3.18 (s, 2H), 2.83 (dd, J=14.0, 6.0 Hz, 1H),2.49-2.46 (m, 1H), 2.40-2.36 (m, 1H), 2.08-1.93 (m, 4H), 1.66-1.60 (m,3H), 1.53-1.49 (m, 3H), 1.32-1.28 (m, 1H), 0.96 (t, J=7.0 Hz, 3H), 0.80(t, J=7.0 Hz, 3H); ESI MS m/z 965 [M+H]⁺.

Example 39 Preparation of 12′-(Propylsulfanyl)vinblastine

A stirred solution of 12′-iodovinblastine (200 mg, 0.214 mmol),1,1′-bis(diphenylphosphino)ferrocene (48 mg, 0.09 mmol),tris(dibenzylideneacetone)dipalladium(0) (20 mg, 0.02 mmol) in NMP (2mL) was deoxygenated with argon for 10 min followed by the addition oftriethylamine (47 μL, 0.428 mmol) and 1-propanethiol (39 μL, 0.428mmol). The reaction mixture was heated to 60° C. for 5 h, cooled to roomtemperature, and then partitioned between methylene chloride and brine.The organic layer was concentrated under reduced pressure and theresidue purified by chromatography (silica, 85:15 MeOH/CH₂Cl₂), thenfurther purified by reverse phase chromatography (C18,acetontrile/water, 0.05% concentrated ammonium hydroxide) to give12′-(propylsulfanyl)vinblastine (36 mg, 19%) as a tan solid: ¹H NMR (300MHz, CDCl₃) δ 8.21 (br s, 1H), 7.57 (s, 1H), 7.29 (dd, J=9, 1 Hz, 1H),7.05 (d, J=9 Hz, 1H), 6.63 (s, 1H), 6.11 (s, 1H), 5.90 (dd, J=10, 4 Hz,1H), 5.49 (d, J=10 Hz, 1H), 5.39 (s, 1H), 4.53 (m, 1H), 4.14 (m, 2H),4.10 (m, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.63 (s, 3H), 3.89-3.56 (m,5H), 3.41 (br s, 1H), 3.30 (d, J=15 Hz, 1H), 3.17 (m, 2H), 2.87 (t, J=7Hz, 4H), 2.82 (m, 2H), 2.70-2.25 (m), 2.08 (s, 3H), 1.82-1.21 (m, 10H),1.05 (m, 6H), 0.72 (t, J=7 Hz, 3H); ESI MS m/z 885 [M+H]⁺.

Example 40 Preparation of 12′-(Ethylsulfanyl)vincristine L-Tartrate

12′-iodovincristine (650 mg, 0.684 mmol),tris(dibenzylideneacetone)dipalladium(0) (94 mg, 0.10 mmol),1,1′-bis(diphenylphosphino)ferrocene (227 mg, 0.410 mmol),1-methyl-2-pyrrolidinone (6 mL), and triethylamine (0.23 mL) werecombined in a resealable glass test tube. Argon was bubbled through thesolution for 10 min, then ethanethiol (0.25 mL, 3.4 mmol) was added, thetest tube sealed, and the mixture was heated to 60° C. After 4 h,additional ethanethiol (0.25 mL, 3.4 mmol) was added, and the mixturewas heated to 60° C. overnight. After cooling, the mixture was dilutedwith ethyl acetate (250 mL), washed with saturated aqueous NH₄Cl (3×20mL), water and brine, then dried (Na₂SO₄) and evaporated to drynessunder vacuum. The residue was purified by reverse phase chromatography(C18, acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(ethylsulfanyl)vincristine trifluoroacetate (130 mg, 17%) as a whitepowder after lyophilization. After conversion to the free base,treatment with 2 equivalents of L-tartaric acid gave12′-(ethylsulfanyl)vincristine as a salt of L-tartrate: ¹H NMR (500 MHz,CD₃OD) δ 8.95 (s, 1H), 7.61 (s, 1H), 7.28-7.26 (m, 2H), 7.22 (dd, J=8.5,1.5 Hz, 1H), 6.90 (s, 1H), 5.92 (dd, J=10.0, 4.5 Hz, 1H), 5.48 (d,J=10.0 Hz, 1H), 5.14 (s, 1H), 4.64-4.59 (m, 2H), 4.42 (s, 4H), 3.40-3.89(m, 4H), 3.80 (d, J=16.0 Hz, 1H), 3.72-3.57 (m, 9H), 3.47 (dd, J=16.0,5.0 Hz, 2H), 3.40-3.38 (m, 1H), 3.33-3.27 (m, 1H), 3.20 (d, J=14.0 Hz,1H), 3.10 (d, J=14.0 Hz, 1H), 3.00 (d, J=16.0 Hz, 1H), 2.88-2.83 (m,4H), 2.47 (d, J=11.5 Hz, 1H), 2.17-2.08 (m, 1H), 2.00 (s, 3H), 1.88-1.79(m, 1H), 1.64-1.40 (m, 7H), 1.21 (t, J=7.5 Hz, 3H), 0.96 (t, J=7.5 Hz,3H), 0.78 (t, J=7.5 Hz, 3H); ESI MS m/z 885 [M+H]⁺.

Example 41 Preparation of 12′-(Ethylsulfanyl)vinblastineTrifluoroacetate

12′-iodovinblastine (100 mg, 0.10 mmol),tris(dibenzylideneacetone)dipalladium(0) (14 mg, 0.015 mmol),1,1′-bis(diphenylphosphino)ferrocene (33 mg, 0.060 mmol),1-methyl-2-pyrrolidinone (1 mL), and triethylamine (34 μL) were combinedin a resealable glass test tube. Argon was bubbled through the solutionfor 10 min, then ethanethiol (37 PL, 0.50 mmol) was added, the test tubesealed, and the mixture was heated to 60° C. After 4 h, additionalethanethiol (75 μL, 1.0 mmol) was added, and the mixture was heated to60° C. overnight. After cooling, the mixture was diluted with ethylacetate (100 mL), washed with saturated aqueous NH₄Cl (3×15 mL), waterand brine, then dried (Na₂SO₄) and evaporated to dryness under vacuum.The residue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(ethylsulfanyl)vinblastine trifluoroacetate (10 mg, 11%) as a whitepowder after lyophilization: ¹H NMR (500 MHz, CD₃OD) δ 9.75 (br s, 1H),7.61 (s, 1H), 7.26-7.21 (m, 2H), 6.70 (s, 1H), 6.42 (s, 1H), 5.95 (dd,J=10.5, 5.0 Hz, 1H), 5.66 (d, J=10.0 Hz, 1H), 5.35 (s, 1H), 4.64 (dd,J=17.5, 11.5, 1H), 3.97-3.91 (m, 3H), 3.86 (s, 3H), 3.82 (s, 3H),3.77-3.57 (m, 8H), 3.49 (d, J=16.0 Hz, 1H), 3.34-3.31 (m, 1H), 3.27-3.16(m, 3H), 2.89 (dd, J=14.5, 6.5 Hz, 1H), 2.85 (q, J=7.5 Hz, 2H), 2.78 (s,3H), 2.46 (dd, J=15.5, 4.5 Hz, 1H), 2.37-2.31 (m, 1H), 2.07 (s, 2H),2.05-1.99 (m, 2H), 1.78-1.50 (m, 6H), 1.39-1.32 (m, 1H), 1.21 (t, J=7.5Hz, 3H), 0.97 (t, J=7.5 Hz, 3H), 0.80 (t, J=7.5 Hz, 3H); ESI MS m/z 871[M+H]⁺.

Example 42 Preparation of 12′-(Methylsulfanyl)vincristineTrifluoroacetate

2′-iodovincristine (85 mg, 0.089 mmol),tris(dibenzylideneacetone)dipalladium(0) (12 mg, 0.013 mmol),1,1′-bis(diphenylphosphino)ferrocene (30 mg, 0.053 mmol),1-methyl-2-pyrrolidinone (1 mL), and triethylamine (31 μL) were combinedin a resealable glass test tube. Argon was bubbled through the solutionfor 10 min, and methanethiol (0.22 mL of a 4 N solution in NMP, 0.89mmol) was added, the test tube sealed, and the mixture was heated to 65°C. After 3h, additional methanethiol (0.22 mL of a 4 N solution in NMP,0.89 mmol) was added, and the mixture was heated to 65° C. overnight.After cooling, the mixture was diluted with ethyl acetate (100 mL),washed with saturated aqueous NH₄Cl (3×15 mL), water and brine, thendried (Na₂SO₄) and evaporated to dryness in vacuo. The residue waspurified by reverse phase chromatography (Cl 8, acetonitrile/water,0.05% trifluoroacetic acid) to provide 12′-(methylsulfanyl)vincristinetrifluoroacetate (9 mg, 9%) as a white powder after lyophilization: ¹HNMR (500 MHz, CD₃OD) δ 9.91 (br s, 1H), 8.99 (s, 1H), 7.55 (s, 1H), 7.34(s, 1H), 7.29 (d, J=8.5 Hz, 1H), 7.19 (d, J=8.5 Hz, 1H), 6.96 (s, 1H),5.98 (dd, J=10.0, 5.0 Hz, 1H), 5.74 (d, J=10.0 Hz, 1H), 5.19 (s, 1H),4.69-4.67 (m, 2H), 4.03-3.76 (m, 11H), 3.73 (s, 3H), 3.67 (s, 3H), 3.52(d, J=16.0 Hz, 1H), 3.36-3.32 (m, 1H), 3.17 (s, 2H), 2.84 (dd, J=14.0,5.5 Hz, 1H), 2.54-2.46 (m, 1H), 2.45 (s, 3H), 2.44-2.36 (m, 1H), 2.05(s, 3H), 2.03-1.93 (m, 1H), 1.68-1.58 (m, 3H), 1.54-1.47 (m, 3H),1.31-1.29 (m, 1H), 0.97 (t, J=7.5 Hz, 3H), 0.83 (t, J=7.5 Hz, 3H); ESIMS m/z 871 [M+H]⁺.

Example 43 Preparation of 12′-(Methylsulfanyl)vinblastineTrifluoroacetate

12′-iodovinblastine (45 mg, 0.048 mmol),tris(dibenzylideneacetone)dipalladium(0) (9.0 mg, 0.0096 mmol),1,1′-bis(diphenylphosphino)ferrocene (21 mg, 0.038 mmol),1-methyl-2-pyrrolidinone (0.5 mL), and triethylamine (17 μL) werecombined in a resealable glass test tube. Argon was bubbled through thesolution for 10 min, then methanethiol was bubbled through for 10 sec,the test tube sealed, and the mixture was heated to 60° C. for 5 h.After cooling, the mixture was diluted with ethyl acetate (75 mL),washed with saturated aqueous NH₄Cl (3×15 mL), water and brine, thendried (Na₂SO₄) and evaporated to dryness under vacuum. The residue waspurified by reverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′-(methylsulfanyl)vinblastinetrifluoroacetate (3.5 mg, 7%) as a white powder after lyophilization: ¹HNMR (500 MHz, CD₃OD) δ 7.54 (s, 1H), 7.24 (d, J=8.5 Hz, 1H), 7.18 (dd,J=8.5, 1.5 Hz, 1H), 6.67 (s, I H), 6.42 (s, 1H), 5.94 (dd, J=10.5, 5.0Hz, 1H), 5.66 (d, J=10.5 Hz, 1H), 5.35 (s, 1H), 4.63 (dd, J=17.0, 11.0Hz, 1H), 3.97-3.90 (m, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 3.79-3.58 (m,8H), 3.49 (d, J=15.5 Hz, 1H), 3.34-3.30 (m, 1H), 3.27-3.16 (m, 3H), 2.89(dd, J=14.5, 6.0 Hz, 1H), 2.78 (s, 3H), 2.53-2.47 (m, 1H), 2.45 (s, 3H),2.38-2.32 (m, 1H), 2.07 (s, 3H), 2.05-2.01 (m, 2H), 1.78-1.64 (m, 3H),1.56-1.50 (m, 3H), 1.39-1.36 (m, 1H), 0.97 (t, J=7.5 Hz, 3H), 0.80 (t,J=7.5 Hz, 3H); ESI MS m/z 857 [M+H]⁺.

Example 44 Preparation of12′-(tert-Butoxycarbonylmethylsulfanyl)vinblastine Trifluoroacetate

tert-Butyl thioglycolate (160 mg, 1.08mmol), 12′-iodovinblastine (101mg, 0.108 mmol), triethylamine (218 mg, 2.15 mmol),tris(dibenzylideneacetone)dipalladium(0) (9.8 mg, 0.011 mmol) and1,1′bis(diphenylphosphino)ferrocene (24 mg, 0.043 mmol) were combined inN-methyl-2-pyrrolidinone (2.5 mL) and the reaction mixture wasdeoxygenated by bubbling argon through the solution for 30 min. Themixture was heated at 60° C. for 2 h then diluted with ethyl acetate (20mL). The organic solution was washed with water (5 mL), saturated NaHCO₃(5 mL) and brine (5 mL), dried over MgSO₄, and evaporated to dryness invacuo. The residue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(tert-butoxycarbonylmethylsulfanyl)vinblastine trifluoroacetate (55mg, 43%), which was a white powder after lyophilization: ¹H NMR (500MHz, CD₃OD) δ 9.80 (br s, 1H), 7.69 (s, 1H), 7.26 (m, 2H), 6.68 (s, 1H),6.42 (s, 1H), 5.94 (dd, J=10.4, 4.2 Hz, 1H), 5.65 (d, J=10.4 Hz, 1H),5.35 (s, 1H), 4.83 (m, 1H), 4.64 (dd, J=17.1, 11.2, 1H), 3.97-3.54 (m,6H), 3.86 (s, 3H), 3.81 (s, 3H), 3.69 (s, 3H), 3.46 (m, 3H), 3.33 (m,1H), 3.21 (m, 1H), 3.18 (s, 2H), 2.88 (m, 1H), 2.77 (s, 3H), 2.47 (dd,J=16.3, 4.7 Hz, 1H), 2.34 (m, 1H), 2.07 (s, 3H), 2.02 (m, 2H), 1.74 (m,1H), 1.66 (m, 2H), 1.54 (m, 3H), 1.38 (s, 9H), 0.97 (t, J=7.4 Hz, 3H),0.79 (t, J=7.2 Hz, 3H); ESI MS m/z 957 [M+H]⁺.

Example 45 Preparation of 12′-(Carboxymethylsulfanyl)vinblastineTrifluoroacetate

12′-tert-Butoxy carbonylmethylsulfanylvinblastine (26 mg, 0.022 mmol)was taken up in methylene chloride (1 mL) and cooled to 0° C.Trifluoroacetic acid (2 mL) was added dropwise and the reaction mixturewas stirred at 0° C. for 20 min then at room temperature for 30 min. Thereaction mixture was evaporated to dryness in vacuo and the residue wastaken up in deionized water (1 mL) and lyophilized to provide12′-(carboxymethylsulfanyl)vinblastine trifluoroacetate (25 mg,quantitative yield) as a white powder: ¹H NMR (500 MHz, CD₃OD) δ 9.77(br s, 1H), 7.71 (s, 1H), 7.28 (m, 2H), 6.69 (s, 1H), 6.42 (s, 1H), 5.94(dd, J=10.3, 4.1 Hz, 1H), 5.65 (d, J=10.5 Hz, 1H), 5.35 (s, 1H), 4.82(m, 1H), 4.65 (dd, J=16.1, 11.4 Hz, 1H), 3.96-3.44 (m, 8H), 3.86 (s,3H), 3.81 (s, 3H), 3.69 (s, 3H), 3.33 (m, 1H), 3.24 (m, 2H), 3.18 (s,2H), 2.88 (dd, J=14.6, 6.1 Hz, 1H), 2.77 (s, 3H), 2.57 (m, 1H), 2.34 (m,1H), 2.07 (s, 3H), 2.04 (m, 2H), 1.72 (m, 1H), 1.65 (m, 2H), 1.53 (m,3H), 1.37 (m, 1H), 0.97 (t, J=7.4 Hz, 3H), 0.79 (t, J=7.2 Hz, 3H); ESIMS m/z 901 [M+H]⁺.

Example 46 Preparation of12′-(Methylaminocarbonylmethylsulfanyl)vinblastine Trifluoroacetate

12′-tert-Butoxy carbonylmethylsulfanylvinblastine (19 mg, 0.016 mmol)was taken up in methylene chloride (I mL) and cooled to 0° C.Trifluoroacetic acid (2 mL) was added dropwise and the reaction mixturewas stirred at 0° C. for 20 min then at room temperature for 30 min. Thereaction mixture was evaporated to dryness in vacuo, taken up in DMF (1mL), then HATU (9 mg, 0.024 mmol), methyl amine (40% aqueous, 14 μL,0.16 mmol), and triethylamine (22 mL, 0.16 mmol) were added. Thenreaction mixture was stirred under nitrogen for 24 h then the solventwas removed in vacuo. The residue was purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid) toprovide 12′-(methylaminocarbonylmethylsulfanyl)vinblastinetrifluoroacetate (1.6 mg, 9%) as a white powder after lyophilization: ¹HNMR (500 MHz, CD₃OD) δ 9.75 (br s, 1H), 7.99 (br s, 1H), 7.68 (s, 1H),7.24 (m, 2H), 6.63 (s, 1H), 6.41 (s, 1H), 5.93 (dd, J=9.8, 3.9 Hz, 1H),5.60 (d, J=9.4 Hz, 1H), 5.35 (s, 1H), 4.86 (m, 1H), 4.62 (m, 1H),3.96-3.54 (m, 6H), 3.85 (s, 3H), 3.81 (s, 3H), 3.68 (s, 3H), 3.51 (s,2H), 3.37 (m, 1H), 3.18 (m, 3H), 3.08 (m, 1H), 2.87 (m, 1H), 2.77 (s,3H), 2.69 (s, 3H), 2.47 (m, 1H), 2.31 (m, 1H), 2.07 (s, 3H), 1.98 (m,2H), 1.72 (m, 1H), 1.66 (m, 2H), 1.52 (m, 3H), 1.31 (m, 1H), 0.97 (t,J=7.4 Hz, 3H), 0.78 (t, J=7.1 Hz, 3H); ESI MS m/z 914 [M+H]⁺.

Example 47 12′-(Methoxycarbonylethylsulfanyl)vincristine

12′-iodovincristine (200 mg, 0.210 mmol),1,1′-bis(diphenylphosphino)ferrocene (47 mg, 0.085 mmol),tris(dibenzylideneacetone)dipalladium(0) (21 mg, 0.023 mmol),triethylamine (0.050 mL, 0.36 mmol), and methyl 3-mercaptopropionate(0.050 mL, 0.45 mmol) were combined in N-methylpyrrolidinone (4 mL) andheated at 60° C. under argon for 16 h. After cooling, the mixture wasdiluted with methylene chloride (10 mL) and washed with brine (10 mL).The aqueous layer was extracted with methylene chloride (2×10 mL), andthe combined organic layers were dried (Na₂SO₄) and evaporated todryness in vacuo. The residue was dissolved in ethyl acetate (50 mL),washed with water (3×20 mL) and brine (20 mL), dried over Na₂SO₄ andevaporated to dryness in vacuo. Purification by reverse phasechromatography (C18, methanol/water) gave12′-(methoxycarbonylethylsulfanyl)vincristine (29.7 mg, 15%) as anoff-white powder: ¹H NMR (300 MHz, CD₃OD) δ 8.93 (s, 1H), 7.83-7.15 (m,4H), 6.93 (s, 1H), 5.89 (dd, J=9.7, 4.9 Hz, 1H), 5.41 (d, J=9.9 Hz, 1H),5.15 (s, 1H), 4.58 (s, 1H), 4.15-3.94 (m, 2H), 3.90 (s, 3H), 3.67 (s,3H), 3.63 (s, 6H), 3.39-3.23 (m, 6H), 3.07-3.01 (m, 4H), 2.90-2.72 (m,3H), 2.62-2.52 (m, 3H), 2.43-2.27 (m, 2H), 2.11-1.95 (m, 4H), 1.80-1.76(m, 1H), 1.52-1.37 (m, 3H), 1.32-1.29 (m, 3H), 0.89 (t, J=7.2 Hz, 3H),0.78 (t, J=7.0 Hz, 3H); ESI MS m/z 943 [M+H]⁺.

Example 48 12′-(2-(N,N-Dimethylamino)ethylsulfanyl)vinblastineTrifluoroacetate

To a mixture of 12′-iodovinblastine (47 mg, 0.05 mmol),tris(dibenzylideneacetone)-dipalladium(0) (4.6 mg, 0.005 mmol),1,1′-bis(diphenylphosphino)ferrocene (11 mg, 0.02 mmol), triethylamine(28 μL, 0.2 mmol) and 1-methyl-2-pyrrolidinone (2 mL) was added2-(dimethylamino)ethanethiol hydrochloride (14.2 mg, 0.1 mmol). Afterthe addition was complete, the reaction mixture was deoxygenated with anargon purge and was then heated to 70° C. for 6 h. An additional 0.5equivalents of all the reagents, except 12-iodovinblastine, were addedto the reaction mixture at room temperature. The resulting mixture wasdeoxygenated again and then heated to 70° C. for an additional 20 h. Thereaction mixture was cooled to room temperature, diluted with water andextracted with CH₂Cl₂ (2×20 mL). The combined organics were washed withwater and brine, and then dried (MgSO₄). Purification by flash columnchromatography (silica, 97:2:1 CH₂Cl₂/CH₃OH/Et₃N) followed bypreparative TLC (silica, 2:3 CH₂Cl₂/CH₃OH) afforded12′-(2-dimethylaminoethylsulfanyl)vinblastine as a tan solid (15 mg,33%). The solid was dissolved in CH₂Cl₂ (1 mL) and treated with a dropof trifluoroacetic acid. The solution was evaporated to give the12′-(2-(N,N-dimethylamino)ethylsulfanyl)vinblastine trifluoroacetate (15mg, 73%): ¹H NMR (300 MHz, CD₃OD) δ 9.84 (s, 1H), 7.67 (s, 1H), 7.22 (s,2H), 6.61 (s, 1H), 6.33 (s, 1H), 5.84 (dd, J=10.5, 3.9 Hz, 1H), 5.58,(d, J=10.5 Hz, 1H), 5.26 (s, 1H), 4.60-4.54 (m, 1H), 3.89-3.81 (m, 3H),3.76 (s, 3H), 3.72 (s, 3H), 3.70 (s, 2H), 3.63 (s, 2H), 3.58 (s, 3H),3.44-3.37 (m, 2H), 3.14-3.09 (m, 5H), 3.03-2.96 (m, 1H), 2.85 (s, 2H),2.83-2.79 (m, 2H), 2.77 (s, 6H), 2.69 (s, 3H), 2.38-2.33 (m, 1H),2.33-2.25 (m, 1H),1.98-1.89 (m, 1H), 1.98 (s, 3H), 1.69-1.61 (m, 1H),1.57-1.56 (m, 2H), 1.51-1.40 (m, 3H), 0.88 (t, J=7.3 Hz, 3H), 0.67 (t,J=7.1 Hz, 3H); ESI MS m/z 914 [M+H]⁺.

Example 49 Preparation of12′-[3-(Morpholin-4-yl)propylsulfanyl]vinblastine Trifluoroacetate

A mixture of 12′-(3-mesyloxypropylsulfanyl)vinblastine (75 mg, 0.077mmol) and morpholine (33.4 μL, 0.38 mmol) in THF (2 mL) was heated toreflux for 24 h and then cooled to room temperature. The reactionmixture was diluted with water and extracted with CH₂Cl₂ (2×20 mL). Thecombined organics were washed with water and brine, and then dried(MgSO₄). Purification by column chromatography (silica, 9:1CH₂Cl₂/CH₃OH) followed by preparative TLC (silica, 4:1 CH₂Cl₂/CH₃OH)gave 12′-[3-(morpholin-4-ylpropylsulfanyl]vinblastine as a solid (29 mg,39%). The solid was dissolved in CH₂Cl₂ (1 mL) and treated with a dropof trifluoroacetic acid. The solution was evaporated to give12′-[3-(morpholin-4-yl)propylsulfanyl]vinblastine trifluoroacetate (33.4mg, 87%): ¹H NMR (300 MHz, CD₃OD) δ 9.83 (s, 1H), 7.71 (s, 1H), 7.29 (s,2H), 6.73 (s, 1H), 6.44 (s, 1H), 5.96 (dd, J=10.1, 4.5 Hz, 1H), 5.68,(d, J=10.4 Hz, 1H), 5.37 (s, 1H), 4.74-4.60 (m, 1H), 4.05-3.67 (m, 13H),3.87 (s, 3H), 3.83 (s, 3H), 3.70 (s, 3H), 3.58-3.05 (m, 11H), 2.96 (t,J=6.7 Hz, 2H), 2.80 (s, 3H), 2.50-2.29 (m, 2H), 2.09 (s, 3H), 2.08-1.96(m, 3H), 1.80-1.67 (m, 3H), 1.61-1.51 (m, 3H), 1.39-1.37 (m, 1H), 0.99(t, J=7.3 Hz, 3H), 0.81 (t, J=7.0 Hz, 3H); ESI MS m/z 970 [M+H]⁺.

Example 50 Preparation of12′-[3-(Piperidin-1-yl)propylsulfanyl]vinblastine Trifluoroacetate

A mixture of 12′-(3-mesyloxypropylsulfanyl)vinblastine (100 mg, 0.102mmol) and piperidine (51 μL, 0.51 mmol) in THF (3 mL) was heated toreflux for 72 h and then cooled to room temperature. The reactionmixture was diluted with water and extracted with CH₂Cl₂ (2×20 mL). Thecombined organics were washed with water and brine, and then dried(MgSO₄). Purification by preparative TLC (silica, 4:1 acetone/CH₃OH)followed by ion exchange chromatography (Isolute SCX-2 column, 3:1MeOH/NH₄OH) gave 12′-[3-(piperidin-1-yl)propylsulfanyl]vinblastine as asolid (37 mg, 37%). The solid was dissolved in CH₂Cl₂ (1 mL) and treatedwith a drop of trifluoroacetic acid. The solution was evaporated to give12′-[3-(piperidin-1-yl)propylsulfanyl]vinblastine trifluoroacetate (45mg, 90%): ¹H NMR (300 MHz, CD₃OD) δ 9.84 (s, 1H), 7.70 (s, 1H), 7.29 (s,2H), 6.73 (s, 1H), 6.44 (s, 1H), 5.96 (dd, J=10.4, 4.3 Hz, 1H), 5.68,(d, J=10.4 Hz, 1H), 5.37 (s, 1H), 4.74-4.60 (m, 1H), 4.01-3.91 (m, 3H),3.87 (s, 3H), 3.83 (s, 3H), 3.75 (s, 1H), 3.70 (s, 3H), 3.68-3.63 (m,1H), 3.56-3.40 (m, 4H), 3.28-3.05 (m, 4H), 2.98-2.87 (m, 4H), 2.80 (s,3H), 2.53-2.30 (m, 2H), 2.09 (s, 3H), 2.08-1.32 (m, 20H), 0.99 (t, J=7.3Hz, 3H), 0.82 (t, J=7.2 Hz, 3H); ESI MS m/z 968 [M+H]⁺.

Example 51 Preparation of12′-[2-Pyrrolidin-1-yl-ethylsulfanyl]vinblastine Trifluoroacetate

To a mixture of 12′-iodovinblastine (176 mg, 0.17 mmol),tris(dibenzylideneacetone)dipalladium(0) (15 mg, 0.016 mmol),1,1′-bis(diphenylphosphino)ferrocene (18 mg, 0.030 mmol), triethylamine(81 μL, 0.59 mmol) and 1-methyl-2-pyrrolidinone (2 mL) was added2-pyrrolidin-1-yl-ethanethiol (28 mg, 0.50 mmol). After the addition wascomplete, the reaction mixture was deoxygenated with an argon purge andwas then heated to 70° C. for 16 h. After cooling to room temperature,the reaction mixture was diluted with water and extracted with EtOAc(2×20 mL). The combined organics were washed with H₂O and brine, andthen dried (Na₂SO₄) and concentrated. Purification by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid)gave 12′-(2-pyrrolidin-1-yl-ethylsulfanyl)vinblastine trifluoroacetate(33 mg, 21%): ¹H NMR (500 MHz, CD₃OD) δ 9.93 (m, 1H), 7.75 (s, 1H), 7.31(s, 2H), 6.71 (s, 1H), 6.42 (s, 1H), 5.93 (dd, J=10.5, 3.0 Hz, 1H), 5.67(dd, J=10.5, 3.0 Hz, 1H), 5.35 (s, 1H), 4.70-4.67 (m, 1H), 3.97-3.90 (m,3H), 3.84 (s, 3H), 3.82-3.80 (m, 4H), 3.75-3.72 (m, 2H), 3.69-3.62 (m,7H), 3.52-3.48 (m, 1H), 3.36-3.27 (m, 3H), 3.19-3.16 (m, 4H), 3.06-3.02(m, 2H), 2.89 (d, J=14.5, 6.0 Hz, 1H), 2.77 (s, 3H), 2.48-2.44 (m, 1H),2.37-2.32 (m, 1H), 2.11-1.98 (m, 6H), 2.07 (s, 3H), 1.77-1.72 (m, 1H),1.65 (d, J=4.0 Hz, 2H), 1.59-1.50 (m, 3H), 1.40-1.36 (m, 1H), 0.97 (t,J=7.5 Hz, 3H), 0.79 (t, J=7.5 Hz, 3H); ESI MS m/z 940 [M+H]⁺.

Example 52 Preparation of 12′-[2-(Acetylamino)ethylsulfanyl]vinblastineTrifluoroacetate

To a mixture of 12′-iodovinblastine (39 mg, 0.04 mmol),tris(dibenzylideneacetone)dipalladium(0) (3.6 mg, 0.004 mmol),1,1′-bis(diphenylphosphino)ferrocene (9.1 mg, 0.016 mmol), triethylamine(12 μL, 0.08 mmol) and 1-methyl-2-pyrrolidinone (1 mL) was addedN-(2-mercaptoethyl)acetamide (9 μL, 0.08 mmol). After the addition wascomplete, the reaction mixture was deoxygenated with an argon purge andwas then heated to 70° C. for 2 h and then cooled to room temperature.The reaction mixture was diluted with water and extracted with CH₂Cl₂(2×20 mL). The combined organics were washed with H₂O and brine, andthen dried (MgSO₄). Purification by preparative TLC (silica, 92.5:7.5CH₂Cl₂/CH₃OH) gave an orange solid. The solid was further purified byprep-TLC (silica gel, CH₃OH/acetone, 4:1) to afford12′-[2-(acetylamino)ethylsulfanyl]vinblastine as off-white solid (19 mg,50%). The solid was dissolved in CH₂Cl₂ (1 mL) and treated with a dropof trifluroacetic acid. The solution was evaporated to give12′-[2-acetylaminoethylsulfanyl]vinblastine trifluoroacetate (19 mg,80%): ¹H NMR (300 MHz, CD₃OD) δ 9.75 (s, 1H), 7.72 (s, 1H), 7.27 (s,2H), 6.72 (s, 1H), 6.44 (s, 1H), 5.96 (dd, J=10.4, 4.3 Hz, 1H), 5.68,(d, J=10.4 Hz, 1H), 5.38 (s, 1H), 4.70-4.61 (m, 1H), 3.98-3.64 (m, 8H),3.87 (s, 3H), 3.84 (s, 3H), 3.71 (s, 3H), 3.60-3.48 (m, 1H), 3.44-3.25(m, 1H), 3.21 (s, 2H), 2.95 (t, J=6.9 Hz, 3H), 2.80 (s, 3H), 2.48 (dd,J=6.2, 4.5 Hz, 1H), 2.42-2.32 (m, 1H), 2.09 (s, 3H), 2.05-1.99 (m, 1H),1.93 (s, 3H), 1.81-1.68 (m, 3H), 1.61-1.48 (m, 3H), 1.42-1.25 (m, 4H),0.99 (t, J =7.5 Hz, 3H), 0.82 (t, J =7.2 Hz, 3H); ESI MS m/z 928 [M+H]⁺.

Example 53 Preparation of 12′-Thiovinblastine Trifluoroacetate

12′-Iodovinblastine (60 mg, 0.064 mmol), thiotriisopropysilyl potassiumsalt (44 mg, 0.192 mmol), and tetrakis(triphenylphosphine) palldium(0)(15 mg, 0.012 mmol) were combined in benzene/tetrahydrofuran (4 mL, 3:1)and the reaction mixture was deoxygenated by bubbling argon through thesolution for 30 min. The mixture was heated at 65° C. for 1.5 h thendiluted with ethyl acetate (15 mL). The organic solution was washed withwater (5 mL), saturated aqueous NaHCO₃ (5 mL) and brine (5 mL), driedover MgSO₄, and evaporated to dryness in vacuo. The residue was purifiedby reverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′thiovinblastine trifluoroacetate(18.9 mg, 28%) which was a white powder after lyophilization: ¹H NMR(500 MHz, CD₃OD) δ 9.62 (br s, 1H), 7.51 (s, 1H), 7.18 (d, J=8.4 Hz,1H), 7.10 (dd, J=8.4, 1.1 Hz, 1H), 6.64 (s, 1H), 6.41 (s, 1H), 5.94 (dd,J=10.3, 4.0 Hz, 1H), 5.63 (d, J=10.5 Hz, 1H), 5.35 (s, 1H), 4.84 (m,1H), 4.61 (dd, J=16.6, 11.2 Hz, 1H), 3.95-3.56 (m, 7H), 3.85 (s, 3H),3.81 (s, 3H), 3.69 (s, 3H), 3.44 (m, 2H), 3.27 (m, 1H), 3.18 (m, 3H),2.88 (dd, J=14.2, 6.0 Hz, 1H), 2.77 (s, 3H), 2.47 (dd, J=15.8, 4.3 Hz,1H), 2.34 (m, 1H), 2.07 (s, 3H), 2.04 (m, 1H), 1.78-1.62 (m, 3H), 1.52(q, J=7.6 Hz, 2H), 1.38 (m, 1H), 0.96 (t, J=7.4 Hz, 3H), 0.79 (t, J=7.3Hz, 3H); ESI MS m/z 843 [M+H]⁺.

Example 54 12′-(3-Hydroxyphenylsulfanyl)vincristine

A solution of 12′-iodovincristine (158 mg, 0.17 mmol),tris(dibenzylideneacetone)dipalladium(0) (18 mg, 0.02 mmol),triethylamine (0.05 mL) in N-methylpyrrolidine (4 mL) was deoxygenatedwith argon for 10 min, then 3-hydroxythiophenol (0.05 mL) was added andthe reaction mixture was heated to 60° C. overnight. After this time,additional tris(dibenzylideneacetone)dipalladium(0) (15 mg) was addedand heating was continued for another 24 h. The reaction mixture wascooled to room temperature and diluted with CH₂Cl₂. The organic layerwas washed with brine, dried (sodium sulfate), filtered, andconcentrated under reduced pressure to give a dark brown solid.Purification by flash column chromatography (silica, 94.5:5:0.5CH₂Cl₂/MeOH/NH₄OH) followed by reverse phase chromatography (C18,MeOH/water) gave 12′-(3-hydroxyphenylsulfide)vincristine (2.8 mg, 2%) asan off-white solid: ¹H NMR (300 MHz, CD₃OD) δ 8.94 (s, 1H), 7.78 (s,1H), 7.30 (d, J=8 Hz, 1H), 7.22 (s, 1H), 7.20 (d, J=8 Hz, 1H), 7.00 (t,J=8 Hz, 1H), 6.94 (s, 1H), 6.53 (d, J=8 Hz, 1H), 6.48 (d, J=8 Hz, 1H),6.45 (s, 1H), 5.91 (dd, J=10, 5 Hz, 1H), 5.43 (d, J=10 Hz, 1H), 5.16 (s,1H), 4.58 (s, 1H), 4.10 (t, J=15 Hz, 1H), 4.00 (m, 1H), 3.90 (s, 3H),3.87 (s, 1H), 3.72 (s, 1H), 3.69 (s, 3H), 3.63 (s, 3H), 3.47-3.13 (m),3.09 (s, 1H), 3.04 (m, 1H), 2.90 (d, J=17 Hz, 1H), 2.81 (m, 2H), 2.67(tm, J=10 Hz, 1H), 2.44 (d, J=11 Hz, 1H), 2.30 (d, J=11 Hz, 1H), 2.09(m, 1H), 2.03 (s, 3H), 1.75 (m, 1H), 1.60-1.48 (m, 3H), 1.34 (q, J=7 Hz,2H), 0.89 (t, J=7 Hz, 3H), 0.79 (t, J=7 Hz, 3H); ESI MS m/z 949 [M+H]⁺.

Example 55 Preparation of 12′-(2-Hydroxyphenylsulfanyl)vinblastine

A solution of iodovinblastine (200 mg, 0.214 mmol),1,1′bis(diphenylphosphino)ferrocene (48 mg, 0.09 mmol),tris(dibenzylideneacetone)dipalladium(0) (20 mg, 0.02 mmol), andtriethylamine (47 μL, 0.43 mmol) in NMP (3.2 mL) was deoxygenated withargon for 10 min, then 2-hydroxythiophenol (44.3 μL, 0.428 mmol) wasadded and the reaction mixture was heated to 60° C. for 22 h. Thereaction mixture was cooled to room temperature and then partitionedbetween methylene chloride and brine. The organic layer was concentratedunder reduced pressure and the resulting residue was purified bychromatography (silica, 85:15 CH₂Cl₂/MeOH). Further purification byreverse phase chromatography (C18, acetonitrile/water, 0.05%concentrated ammonium hydroxide) gave12′-(2-hydroxyphenylsulfanyl)vinblastine (28 mg, 8%) as a white solid:¹H NMR (300 MHz, DMSO-d₆) δ 7.76 (s, 1H), 7.38 (d, J=8 Hz, 1H), 7.13 (d,J=8 Hz, 1H), 6.97 (td, J=7, 2 Hz, 1H), 6.82 (d, J=8 Hz, 1H), 6.67 (m,3H), 6.42 (s, 1H), 5.83 (dd, J=10, 4 Hz, 1H), 5.40 (m, 1H), 5.12 (s,2H), 4.37 (m, 1H), 3.90 (s, 3H), 3.82 (t, J=15 Hz, 1H), 3.80-3.20 (m),3.78 (s, 3H), 3.71 (s, 3H), 3.01 (m, 2H), 2.65 (m, 1H), 2.64 (s, 3H),2.20 (m, 2H), 2.02 (m, 1H), 2.01 (s, 3H), 1.73 (m, 1H), 1.68-1.30 (m,5H), 1.15 (m, 1H), 0.85 (t, J=7 Hz, 3H), 0.65 (t, J=7 Hz, 3H); ESI MSm/z 935 [M+H]⁺.

Example 56 Preparation of 12′-(2-Chlorophenylsulfanyl)vincristineTrifluoroacetate

A solution of 12′-iodovincristine (60 mg, 0.05 mmol) in NMP (1.5 mL) wasdeoxygenated with argon for 10 minutes. The reaction vessel was chargedwith 1,1′-bis(diphenylphosphino)ferrocene (20 mg, 0.035 mmol),tris(dibenzylideneacetone)dipalladium(0) (9 mg, 0.009 mmol) and Et₃N (13mg, 0.13 mmol). The mixture was stirred for 20 min at room temperature,2-chlorobenzene-thiol (15 mg, 0.105 mmol) was added and then stirred at60° C. overnight. The mixture was cooled to room temperature, dilutedwith ethyl acetate (100 mL) and washed with saturated aqueous NH₄Cl(3×10 mL) and brine (3×10 mL). The organic layer was dried (Na₂SO₄),filtered and concentrated. The residue was purified by flashchromatography (silica, 10:1 CH₂Cl₂/MeOH) and then by reverse phasechromatography (C-18, acetonitrile/water, 0.05% trifluoroacetic acid) togive 11′-(2-chlorophenylsulfanyl)vincristine (18 mg, 35%)trifluoroacetate: ¹H NMR (500 MHz, CD₃OD) δ 10.25 (s, 1H), 9.00 (s, 1H),7.81 (s, 1H), 7.44 (d, J=8.5 Hz, 1H), 7.36 (s, 1H), 7.34 (dd, J=7.5, 1.0Hz, 1H), 7.28 (dd, J=8.5, 1.5 Hz, 1H), 7.07-6.99 (m, 3H), 6.66 (dd,J=8.0, 1.5 Hz, 1H), 5.99 (dd, J=10.0, 4.5 Hz, 1H), 5.75 (d, J=10.5 Hz,1H), 5.21 (s, 1H), 4.69 (t, J=11.5 Hz, 2H), 4.14 (s, 1H), 4.02-3.98 (m,2H), 3.96 (s, 3H), 3.92-3.82 (m, 2H), 3.75 (s, 3H), 3.68 (s, 3H),3.64-3.54 (m, 3H), 3.38-3.33 (m, 2H), 3.16 (s, 2H), 2.88 (dd, J=14.5,6.0 Hz, 1H), 2.50 (d, J=12.0 Hz, 1H), 2.43-2.38 (m, 1H), 2.05 (s, 3H),2.01-1.96 (m, 1H), 1.68-1.55 (m, 4 H), 1.51 (q, J=7.5 Hz, 2H), 1.33-1.31(m, 1H), 0.96 (t, J=7.5Hz, 3H), 0.84 (t, J=7.0 Hz, 3H); ESI MS m/z 967[M+H]⁺.

Example 57 Preparation of 12′-(Methyldisulfanyl)vinblastineTrifluoroacetate

12′-sulfanylvinblastine (26 mg, 0.031 mmol) andN-(methylthio)phthalimide (12 mg, 0.062 mmol) were combine in benzene (1ml) under nitrogen at room temperature. After stirring for 45 min, thesolvent was removed in vacuo and the residue was purified by reversephase chromatography (C18, acetonitrile/water, 0.05% trifluoroaceticacid) to provide 12′-(methyldisulfanyl)vinblastine as a salt ofthrifluoroacetic acid (11.9 mg, 35%) which was a white powder afterlyophilization: ¹H NMR (500 MHz, CD₃OD) δ 9.86 (br s, 1H), 7.74 (s, 1H),7.34 (dd, J=8.5, 1.5 Hz, 1H), 7.30 (d, J=8.5 Hz, 1H), 6.69 (s, 1H), 6.42(s, 1H), 5.94 (dd, J=10.4, 5.3 Hz, 1H), 5.66 (d, J=10.5 Hz, 1H), 5.35(s, 1H), 4.96-3.61 (m, 7H), 4.84 (m, 1H), 4.66 (dd, J=17.2, 11.1 Hz,1H), 3.86 (s, 3H), 3.81 (s, 3H), 3.70 (s, 3H), 3.49 (d, J=15.8 Hz, 1H),3.34 (m, 1H), 3.24 (m, 1H), 3.18 (s, 1H), 2.87 (dd, J=14.4, 6.1 Hz, 1H),2.78 (s, 3H), 2.47 (m, 1H), 2.41 (s, 3H), 2.34 (m, 1H), 2.07 (s, 3H),2.03 (m, 2H), 1.73 (m, 1H), 1.65 (m, 2H), 1.57 (m, 1H), 1.52 (q, J=7.6Hz, 2H), 1.38 (m, 1H), 0.97 (t, J=7.4 Hz, 3H), 0.81 (t, J=7.2 Hz, 3H);ESI MS m/z 889 [M+H]⁺.

Example 58 Preparation of 12′-(Isopropyldisulfanyl)vinblastineTrifluoroacetate

12′-sulfanylvinblastine (17 mg, 0.020 mmol) and diethylN-isopropylsulfenylhydrazodicarboxylate (15 mg, 0.060 mmol) werecombined in benzene (1 ml) under nitrogen and stirred at roomtemperature for 5 h then at 50° C. overnight. The solvent was removed invacuo and the non basic impurities were removed by flushing the residuethrough an Absolute SCX-2 column, first with methanol then with 10%aqueous ammonium hydroxide/methanol. The basic methanol fractions wereevaporated in vacuo and the residue was purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid) toprovide di(12′-vinblastine)disulfide trifluoroacetate (11.5 mg, 51%) and12′-(isopropyldisulfanyl)vinblastine (3.0 mg, 13%) as white powdersafter lyophilization. The data for the trifluoroacetate of12′-isopropyldisulfanylvinblastine as as follows: ¹H NMR (500 MHz,CD₃OD) δ 7.73 (s, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.27 (d, J=8.4 Hz, 1H),6.64 (s, 1H), 6.41 (s, 1H), 5.93 (dd, J=10.7, 5.2 Hz, 1H), 5.59 (d,J=9.5 Hz, 1H), 5.35 (s, 1H), 4.86 (m, 1H), 4.62 (m, 1H), 3.96-3.57 (m,6H), 3.85 (s, 3H), 3.81 (s, 3H), 3.69 (s, 3H), 3.34 (m, 2H), 3.18 (m,3H), 3.04 (m, 1H), 2.90 (m, 1H), 2.77 (s, 3H), 2.71 (m, 1H), 2.49 (dd,J=12.5, 6.2 Hz, 1H), 2.28 (m, 1H), 2.07 (s, 3H), 2.06 (m, 1H), 1.96 (m,1H), 1.74 (m, 1H), 1.66 (m, 2H), 1.52 (m, 3H), 1.38 (m, 1H), 1.28 (d,J=6.7 Hz, 6H), 0.97 (t, J=7.2 Hz, 3H), 0.79 (t, J=7.3 Hz, 3H); ESI MSm/z 917 [M+H]⁺.

Example 59 Preparation of 12′-(tert-Butyldisulfanyl)vinblastineTrifluoroacetate

12′-sulfanylvinblastine (19 mg, 0.023 mmol) and diethylN-tert-butylsulfenylhydrazodicarboxylate (12 mg, 0.045 mmol) werecombined in benzene (1 ml) under nitrogen and stirred at 50° C.overnight. Additional diethyl N-tert-butylsulfenylhydrazodicarboxylate(30 mg, 0.113 mmol) was added and the reaction mixture was kept at 50°C. for 4 d. The solvent was removed in vacuo and the non-basicimpurities were removed by flushing the residue through an AbsoluteSCX-2 column, first with methanol then with 10% aqueous ammoniumhydroxide/methanol. The basic methanol fractions were evaporated invacuo and the residue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluroacetic acid) to providedi(12′-vinblastine)disulfide as a triacetic acid salt (6.2 mg, 33%) and12′-(tert-butyldisulfanyl)vinblastine as a trifluroacetic acid salt(1.9mg, 7.3%). The data for the trifluoroacetate of12′-(tert-butyldisulfanyl)vinblastine is as follows: ¹H NMR (500 MHz,CD₃OD) δ 9.77 (br s, 1H), 7.73 (s, 1H), 7.36 (dd, J=8.5, 1.6 Hz, 1H),7.25 (d, J=8.5 Hz, 1H), 6.68 (s, 1H), 6.42 (s, 1H), 5.94 (dd, J=10.5,4.4 Hz, 1H), 5.64 (d, J=9.5 Hz, 1H), 5.36 (s, 1H), 4.84 (m, 1H), 4.63(dd, J=16.7, 10.5 Hz, 1H), 3.97-3.55 (m, 7H), 3.86 (s, 3H), 3.81 (s,3H), 3.69 (s, 3H), 3.45 (m, 1H), 3.39 (m, 1H), 3.18 (m, 3H), 2.89 (dd,J=14.5, 5.7 Hz, 1H), 2.78 (s, 3H), 2.45 (m, 1H), 2.34 (m, 1H), 2.07 (s,3H), 2.00 (m, 1H), 1.74 (m, 1H), 1.66 (m, 2H), 1.56 (m, 1H), 1.52 (q,J=7.6 Hz, 2H), 1.38 (m, 1H), 1.29 (s, 9H), 0.97 (t, J=7.4 Hz, 3H), 0.79(t, J=7.3 Hz, 3H); ESI MS m/z 931 [M+H]⁺.

Example 60 Preparation of Di-(12′-vinblastine)disulfide Trifluoroacetate

12′-sulfanylvinblastine (17 mg, 0.020 mmol) and diethylN-isopropylsulfenylhydrazodicarboxylate (15 mg, 0.060 mmol) werecombined in benzene (1 ml) under nitrogen and stirred at roomtemperature for 5 h then at 50° C. overnight. The solvent was removed invacuo and the non-basic impurities were removed by flushing the residuethrough an Absolute SCX-2 column, first with methanol then with 10%aqueous ammonium hydroxide/methanol. The basic methanol fractions wereevaporated in vacuo and the residue was purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid) toprovide di(12′-vinblastine)disulfide trifluoroacetate (11.5 mg, 51%) and12′-isopropyldisulfanylvinblastine as a salt of trifluroacetic acid (3.0mg, 13% yield. The data for the trifluoroacetate ofdi-(12′-vinblastine)disulfide is as follows: ¹H NMR (500 MHz, CD₃OD) δ10.00 (br s, 2H), 7.47 (s, 2H), 7.26 (m, 4H), 6.71 (s, 2H), 6.42 (s,2H), 5.96 (dd, J=11.5, 5.3 Hz, 2H), 5.67 (d, J=10.5 Hz, 2H), 5.35 (s,2H), 4.85 (m, 2H), 4.62 (dd, J=16.1, 11.0, 2H), 3.97-3.68 (m, 10 H),3.86 (s, 6H), 3.81 (s, 6H), 3.73 (s, 6H), 3.48 (m, 6H), 3.28-3.08 (m,8H), 2.88 (dd, J=14.6, 6.3 Hz, 2H), 2.78 (s, 6H), 2.46 (dd, J=16.1, 4.7Hz, 2H), 2.34 (m, 2H), 2.08 (s, 6H), 2.01 (m, 2H), 1.72 (m, 2H), 1.66(m, 4H), 1.58 (m, 2H), 1.53 (q, J=7.4 Hz, 4H), 1.38 (m, 2H), 1.00 (t,J=7.4 Hz, 6H), 0.78 (t, J=7.2 Hz, 6H); ESI MS m/z 1683 [M+H]⁺.

Example 61 Preparation of 12′-Formylvinblastine Trifluoroacetate

Vinblastine (55 mg, 0.068 mmole) in trifluoroacetic acid (12 mL) wasadded via pipet to solid hexamethylenetetramine (114 mg, 0.814 mmol)then heated to reflux for 20 min. After cooling to room temperature thereaction mixture was added carefully to a magnetically stirred solutionof saturated aqueous NaHCO₃:water (1:1, 100 mL). Solid NaHCO₃ was thenadded carefully in small portions until no gas evolution was noted. Themixture was extracted with chloroform (3×25 mL) and the combined organicextracts were washed with brine (25 mL), dried over MgSO₄, thenevaporated to dryness in vacuo. The residue was purified by reversephase chromatography (C18, acetonitrile/water, 0.05% trifluoroaceticacid) to provide 12′-formylvinblastine trifluoroacetate (28 mg, 40%): ¹HNMR (500 MHz, CD₃OD) δ 9.95 (s, 1H), 9.66 (s, 1H), 7.59 (s, 1H), 7.28(d, J=8.4 Hz, 1H), 7.20 (d, J=8.3 Hz, 1H), 6.72 (s, 1H), 6.42 (s, 1H),5.94 (dd, J=10.5, 5.1 Hz, 1H), 5.66 (d, J=10.1 Hz, 1H), 5.43 (s, 1H),5.36 (s, 1H), 4.66 (dd, J=17.4, 11.2, 1H), 3.97-3.59 (m, 6H), 3.86 (s,3H), 3.82 (s, 3H), 3.69 (s, 3H), 3.48 (d, J=15.8 Hz, 1H), 3.33 (m, 1H),3.20 (m, 3H), 2.89 (dd, J=14.3, 6.2 Hz, 1H), 2.78 (s, 3H), 2.47 (dd,J=16.2, 5.0 Hz, 1H), 2.35 (m, 1H), 2.07 (s, 3H), 2.03 (m, 2H), 1.75 (m,1H), 1.66 (m, 2H), 1.52 (m, 3H), 1.39 (m, 1H), 0.97 (t, J=7.4 Hz, 3H),0.81 (t, J=7.2 Hz, 3H); ESI MS m/z 839 [M+H]⁺.

Example 62 Preparation of 12′-Formylvincristine Trifluoroacetate

A solution of 12′-iodovincristine (58 mg, 0.062 mmole) in THF (1 mL) wasdeoxygenated with argon for 3 minutes. The reaction vessel was chargedwith tetrakis(triphenylphosphine)palladium(0) (1 mg, 0.011 mmol), andthe flask was sealed with a septum. CO gas (1 atm, balloon) was bubbledinto the solution for 1 minute to generate a saturated solution and toestablish a CO atmosphere in the flask. The reaction mixture was heatedto 50° C., and stirred as a solution of tri-n-butyltin hydride (18 μl,0.068 mmol) in deoxygenated THF (0.5 mL) was slowly added over 30minutes (syringe pump). After the addition was complete, the mixture wasstirred another 15 minutes at 50° C., and then the reaction's porgresswas checked by ESI mass spectral analysis. Starting material wasobserved by ESI MS therefore another 8 μl of tri-n-butyltin hydride indeoxygenated THF (0.5 mL) was added over 15 min. After 30 min, nostarting material remained by ESI mass. The reaction mixture was cool toroom temperature, diluted with diethyl ether (75 mL), and washed with 1N HCl (3×25 mL). The combined aqueous washes were back-extracted withdiethyl ether (25 mL). The acidic water layer was partially neutralizedwith saturated aqueous NaHCO₃ (10 mL), and the resulting turbid mixturewas extracted with EtOAc (3×25 mL). The combined organic extracts weredried (Na₂SO₄) and concentrated. The residue was purified by reversephase chromatography (C18, acetonitrile/water, 0.05% trifluoroaceticacid) to provide 12′-formylvincristine trifluoroacetate (16 mg, 30%): ¹HNMR (500 MHz, CD₃OD) δ 9.93 (br s, 1H), 8.97 (s, 1H), 7.59 (s, 1H),7.35-7.32 (m, 2H), 7.22-7.19 (m, 1H), 6.99 (s, 1H), 5.98-5.95 (m, 1H),5.75-5.72 (m, 1H), 5.43 (s, 1H), 5.16 (s, 1H), 4.71-4.66 (m, 2H),4.04-3.88 (m, 5H), 3.85-3.67 (m, 7H), 3.63-3.57 (m, 1H), 3.52-3.45 (m,1H), 3.23-3.15 (m, 2H), 2.88-2.84 (m, 1H), 2.54-2.49 (m, 1H), 2.43-2.38(m, 1H), 2.08-2.03 (m, 8H), 1.99-1.94 (m, 1H), 1.69-1.62 (m, 3H),1.55-1.49 (m, 3H), 1.35-1.30 (m, 1H), 0.97 (t, J=7.5 Hz, 3H), 083 (t,J=7.0 Hz, 3H); ESI MS m/z 853 [M+H]⁺.

Example 63 Preparation of 12′-(Hydroxymethyl)vinblastine

An ice-cold solution of 12′-formylvinblastine (25 mg, 0.030 mmol) in THF(1 mL) was treated with lithium tri-tert-butoxyaluminum hydride (11 mg,0.045 mmol). The resulting mixture was allowed to warm to roomtemperature, and after stirring for two hours, the reaction was checkedby ESI mass spectral analysis. The reaction was treated again withlithium tri-tert-butoxyaluminum hydride (2×15 mg) over a 20 hour period.The reaction was then quenched with saturated aqueous ammonium chloride(5 mL), diluted with water (7 mL) and extracted with methylene chloride(3×10 mL). The combined extracts were dried (Na₂SO₄) and concentrated toa white solid. Purification by preparative TLC (silica, 7:3EtOAc/hexanes) followed by lyophilization from acetonitrile and watergave 12′-(Hydroxymethyl)vinblastine (15 mg, 60%) as a white solid: ¹HNMR (500 MHz, CDCl₃) δ 9.80 (s, 1H), 8.05 (s, 1H), 7.51 (s, 1H), 7.18(dd, J=1.2, 8.6 Hz, 1H), 7.10 (d, J=4.3 Hz, 1H), 6.59 (s, 1H), 6.10 (s,1H), 5.85 (dd, J=10.1, 3.7 Hz, 1H), 5.46 (s, 1H), 5.30 (d, J=10.2 Hz,1H), 4.77 (s, 2H), 3.95 (t, J=14.2 Hz, 1H), 3.79 (s, 6H), 3.85-3.67 (m,1H), 3.61 (s, 3H), 3.47-3.23 (m, 4H), 3.20-3.05 (m, 2H), 2.90-2.77 (m,3H), 2.71 (s, 3H), 2.65 (s, 1H), 2.52-2.36 (m, 2H), 2.29 (d, J=12.4 Hz,1H), 2.19-2.13 (m, 1H), 2.10 (s, 3H), 1.91-1.55 (m, 5H), 1.52-1.18 (m,6H), 0.89 (t, J=7.5 Hz, 3H), 0.80 (t, J=7.2 Hz, 3H); ESI MS m/z 841[M+H]⁺.

Example 64 Preparation of 12′-(N-Isopropylaminomethyl)vinblastineTrifluoroacetate

A solution of 12′-formylvinblastine (30 mg, 0.036 mmol) in1,2-dichloroethane (1 mL) was treated with isopropylamine (4.2 mg, 0.072mmol) and sodium triacetoxyborohydride (15 mg, 0.072 mmol). Theresulting mixture was stirred at room temperature, until ESI massspectral analysis indicated no starting material remained. The reactionwas quenched with saturated aqueous sodium bicarbonate (5 mL), and theresulting mixture was extracted with methylene chloride (2×10 mL). Thecombined organics were dried (Na₂SO₄) and concentrated to a tan solid.Purification by reverse phase chromatography (C18, acetonitrile/water,0.05% trifluoroacetic acid) gave 12′-(N-isopropylaminomethyl)vinblastinetrifluoroacetate (26.3 mg, 59%): ¹H NMR (500 MHz, CD₃OD) δ 9.87 (s, 1H),7.66 (s, 1H), 7.37 (d, J=8.3 Hz, 1H), 7.24 (dd, J=8.3, 1.4 Hz, 1H), 6.64(s, 1H), 6.40 (s, 1H), 5.91 (dd, J=10.6, 4.2 Hz, 1H), 5.57 (d, J=8.9 Hz,1H), 5.34 (s, 1H), 4.74-4.62 (m, 1H), 4.27 (d, J=3.2 Hz, 2H), 3.99-3.88(m, 2H), 3.87-3.73 (m, 8H), 3.72-3.54 (m, 8H), 3.45-3.34 (m, 2H), 3.19(s, 2H), 3.08-2.94 (m, 1H), 2.89 (dd, J=14.3, 5.9 Hz, 1H), 2.77 (s, 3H),2.47 (dd, J=15.9, 4.6 Hz, 1H), 2.32-2.21 (m, 1H), 2.06 (s, 3H),2.00-1.89 (m, 1H), 1.75-1.70 (m, 1H), 1.66 (d, J=4.1 Hz, 2H), 1.59-1.48(m, 3H), 1.45-1.33 (m, 2H), 1.39 (d, J=6.5 Hz, 6H), 0.97 (t, J=7.5 Hz,3H), 0.77 (t, J=7.4 Hz, 3H); ESI MS m/z 882 [M+H]⁺.

Example 65 Preparation of 12′-Cyanovinblastine

A solution of 12′-iodovinblastine (496 mg, 0.53 mmol) in DMF (15 mL) waspurged with argon for 10 min. Zinc cyanide (137 mg, 1.17 mmol),1,1′-bis(diphenylphosphino)ferrocene (49 mg, 0.05 mmol), andtris(dibenzylideneacetone)dipalladium(0) (59 mg, 0.11 mmol) were added.The reaction mixture was purged with argon again and then heated to 65°C. for 5 h. The reaction mixture was cooled to 0° C., diluted withEtOAc, washed with 5% LiCl, and brine, dried (sodium sulfate), filtered,and concentrated under reduced pressure. Purification by flash columnchromatography (silica, 96:4 to 94:6 CH₂Cl₂/MeOH) gave12′-cyanovinblastine (167 mg, 38%) as a pale yellow solid: ¹H NMR (300MHz, CD₃OD) δ 7.88 (s, 1H), 7.33 (s, 2H), 6.55 (s, 1H), 6.32 (s, 1H),5.82 (dd, J=9, 4 Hz, 1H), 5.35 (m, 2H), 4.06 (m, 2H), 3.81 (s, 3H), 3.76(s, 3H), 3.64 (s, 3H), 3.59 (s, 1H), 3.23 (m), 3.06 (d, J=14 Hz, 1H),2.81 (m, 4H), 2.71 (s, 3H), 2.48 (br d, J=9 Hz, 2H), 2.29 (d, J=14 Hz,1H), 2.03 (m, 4H), 1.86 (m, 1H), 1.63 (m, 1H), 1.53 (m, 2H), 1.40 (m,1H), 1.31 (m, 3H), 0.90 (m, 3H), 0.73 (m, 3H); ESI MS m/z 836 [M+H]⁺.

Example 66 Preparation of 12′-Cyanovincristine

To a solution of iodovincristine (81.5 mg, 0.086 mmol) in DMF (4 mL),purged with argon, was added zinc cyanide (22 mg, 0.19 mmol) followed by1,1′-bis(diphenylphosphino)ferrocene (10.5 mg, 0.019 mmol) andtris(dibenzylideneacetone)dipalladium(0) (8.5 mg, 0.009 mmol). Thereaction mixture was heated at 65° C. for 4.5 h, cooled to roomtemperature, and then partitioned between methylene chloride andsaturated aqueous sodium bicarbonate. The organic layer was dried(sodium sulfate), filtered, and concentrated under reduced pressure togive a brown residue. Purification by flash column chromatography(silica, 97:3 CH₂Cl₂/MeOH) followed by reversed phase chromatography(C18, water/MeOH) gave 12′-cyanovincristine (2.8 mg, 4%) as a whitesolid: ¹H NMR (300 MHz, CD₃OD) δ 8.95 (s, 1H), 7.89 (s, 1H), 7.39 (d,J=8 Hz, 1H), 7.33 (d, J=8 Hz, 1H), 7.22 (s, 1H), 6.86 (s, 1H), 5.89 (dd,J=10, 5 Hz, 1H), 5.41 (d, J=10 Hz, 1H), 5.17 (s, 1H), 4.57 (s, 1H), 4.11(t, J=14 Hz, 1H), 4.00 (m, 1H), 3.95 (s, 3H), 3.87 (s, 1H), 3.72 (s,1H), 3.68 (s, 3H), 3.63 (s, 3H), 3.40-3.10 (m), 3.07 (s, 1H), 3.04 (m,1H), 2.87 (d, J=15 Hz, 1H), 2.79 (m, 2H), 2.63 (td, J=14, 3 Hz, 1H),2.44 (dd, J=15, 3 Hz, 1H), 2.30 (d, J=11 Hz, 1H), 2.05 (m, 2H), 2.00 (s,3H), 1.75 (td, J=12, 7 Hz, 1H), 1.50 (m, 3H), 1.38 (m, 1H), 1.31 (q, J=7Hz, 2H), 0.89 (t, J=7 Hz, 3H), 0.73 (t, J=7 Hz, 3H); ESI MS m/z 850[M+H]⁺.

Example 67 Preparation of 12′-(Methylcarbonyl)vinblastineTrifluoroacetate

Carbon monoxide was bubbled through a solution of 12′-iodovinblastine(81 mg, 0.086 mmol), triethylamine (87 mg, 0.86 mmol) andbis(triphenylphosphine)palladium(II) dichloride (12 mg, 0.017 mmol) in amixture of DMF/methanol (3 mL, 1:1) for 5 min. The reaction mixture washeated at 50° C. for 14 h under one atmosphere of carbon monoxide(balloon). The solution then was diluted with ethyl acetate (20 mL) thenwashed with saturated aqueous NaHCO₃ (2×5 mL) and brine (5 mL), driedover MgSO₄, and evaporated to dryness in vacuo. The residue was purifiedby reverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′-(methylcarbonyl)vinblastinetrifluoroacetate (53 mg, 56%) as a white powder after lyophilization: ¹HNMR (500 MHz, CD₃OD) δ 10.13 (br s, 1H), 8.28 (s, 1H), 7.81 (dd, J=8.7,1.4 Hz, 1H), 7.34 (d, J=8.6 Hz, 1H), 6.67 (s, 1H), 6.42 (s, 1H), 5.93(dd, J=10.5, 4.5 Hz, 1H), 5.66 (d, J=10.5 Hz, 1H), 5.35 (s, 1H), 4.83(m, 1H), 4.71 (dd, J=17.2, 10.9, 1H), 3.98-3.50 (m, 6H), 3.90 (s, 3H),3.86 (s, 3H), 3.81 (s, 3H), 3.70 (s, 3H), 3.46 (d, J=16.2 Hz, 1H), 3.40(dd, J=17.4, 8.4 Hz, 1H), 3.21 (m, 1H), 3.20 (s, 2H), 2.90 (dd, J=14.4,6.1 Hz, 1H), 2.78 (s, 3H), 2.48 (dd, J=16.2, 4.9 Hz, 1H), 2.33 (m, 1H),2.07 (s, 3H), 2.00 (m, 2H), 1.72 (m, 1H), 1.67 (m, 2H), 1.57 (m, 1H),1.53 (q, J=7.5 Hz, 2H), 1.38 (m, 1H), 0.98 (t, J=7.4 Hz, 3H), 0.86 (t,J=7.2 Hz, 3H); ESI MS m/z 869 [M+H]⁺.

Example 68 Preparation of 12′-(2,2,2-Trichloroethylcarbonyl)vinblastineTrifluoroacetate

Carbon monoxide was bubbled through a solution of 12′-iodovinblastine(53 mg, 0.057 mmol), triethylamine (57 mg, 0.565 mmol) andbis(triphenylphosphine)palladium (II) dichloride (8 mg, 0.011 mmol) in amixture of DMF/2,2,2-trichloroethanol (2 mL, 1:1) for 5 min, then thereaction mixture was heated at 50° C. for 9 h under one atmosphere ofcarbon monoxide (balloon). The solution was diluted with ethyl acetate(15 mL) then washed with saturated aqueous NaHCO₃ (2×5 mL) and brine (5mL), dried over MgSO₄ and evaporated to dryness in vacuo. The residuewas purified by reverse phase chromatography (C18, acetonitrile/water,0.05% trifluoroacetic acid) to provide12′-(2,2,2-trichloroethylcarbonyl)vinblastine trifluoroacetate (33 mg,48%) as a white powder after lyophilization: ¹H NMR (500 MHz, CD₃OD) δ10.25 (br s, 1H), 8.36 (s, 1H), 7.90 (dd, J=8.8, 1.5 Hz, 1H), 7.39 (d,J=8.7 Hz, 1H), 6.64 (s, 1H), 6.42 (s, 1H), 5.93 (dd, J=10.4, 4.2 Hz,1H), 5.64 (d, J=10.5 Hz, 1H), 5.35 (s, 1H), 5.08 (d, J=12.2 Hz, 1H),5.05 (d, J=12.1 Hz, 1H), 4.84 (m, 1H), 4.71 (dd, J=17.0, 11.3 Hz, 1H),3.98-3.62 (m, 6H), 3.87 (s, 3H), 3.81 (s, 3H), 3.71 (s, 3H), 3.41 (m,2H), 3.33 (m, 1H), 3.20 (s, 2H), 3.16 (m, 1H), 2.90 (dd, J=14.3, 6.0 Hz,1H), 2.78 (s, 3H), 2.49 (dd, J =16.0, 3.6 Hz, 1H), 2.31 (m, 1H), 2.07(s, 3H), 1.97 (m, 1H), 1.74 (m, 1H), 1.66 (m, 2H), 1.58 (m, 1H), 1.53(q, J=7.5 Hz, 2H), 1.38 (m, 1H), 0.97 (t, J=7.4 Hz, 3H), 0.78 (t, J=7.2Hz, 3H); ESI MS m/z 985 [M+H]⁺.

Example 69 Preparation of 12′-(N-Methylaminocarbonyl)vinblastineTrifluoroacetate

Diisopropylethyl amine (36 mg, 0.276 mmol) was added to12′-(carboxy)vinblastine (15 mg, 0.014 mmol) HATU (10 mg, 0.028 mmol),and N-methylamine (2.0 M in THF, 35 μL, 0.069 mmol) in DMF (0.6 mL).After stirring overnight only the 1-hydroxy-7-azabenztriazole ester wasdetected by ESI MS. N-Methylamine (40% aqueous solution, 0.5 mL) wasadded to the reaction mixture and the solvent was removed in vacuo. Thisprocedure was repeated and the residue was then diluted with ethylacetate (15 mL), washed with saturated aqueous NaHCO₃ (2×5 mL) and brine(5 mL), dried over MgSO₄, and evaporated to dryness in vacuo. Theresidue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(N-methylamino)vinblastine trifluoroacetate (3.4 mg, 22%): ¹H NMR(500 MHz, CD₃OD) δ 10.01 (br s, 1H), 8.07 (s, 1H), 7.64 (dd, J=8.5, 1.5Hz, 1H), 7.33 (d, J=8.5 Hz, 1H), 6.65 (s, 1H), 6.42 (s, 1H), 5.93 (dd,J=10.8, 4.7 Hz, 1H), 5.64 (d, J=10.3 Hz, 1H), 5.36 (s, 1H), 4.85 (m,1H), 4.67 (dd, J=16.7, 11.8 Hz, 1H), 3.98-3.63 (m, 8H), 3.86 (s, 3H),3.81 (s, 3H), 3.69 (s, 3H), 3.38 (m, 2H), 3.19 (m, 3H), 2.94 (s, 3H),2.92 (m, 1H), 2.78 (s, 3H), 2.48 (dd, J =15.9, 4.2 Hz, 1H), 2.35 (m,1H), 2.07 (s, 3H), 2.00 (m, 1H), 1.75 (m, 1H), 1.67 (m, 2H), 1.57 (m,1H), 1.52 (q, J=7.6 Hz, 2H), 1.41 (m, 1H), 0.98 (t, J=7.4 Hz, 3H), 0.79(t, J=7.1 Hz, 3H); ESI MS m/z 868 [M+H]⁺.

Example 70 Preparation of 12′-Acetylvinblastine

A solution of 12′-(trimethylsilylethynyl)vinblastine (130 mg, 0.143mmol) in formic acid (5 mL) was heated at 80° C. for 2 h. After cooling,the mixture was diluted with dichloromethane (25 mL) and poured slowlyinto saturated aqueous NaHCO₃. The organic layer was washed with brine,dried (Na₂SO₄), and concentrated under reduced pressure. Purification byflash chromatography (silica, 98:2:0.5 to 96:4:0.5CHCl₃/MeOH/triethylamine) gave 12′-acetylvinblastine (88 mg, 84%). ¹HNMR (300 MHz, CD₃OD) 8.19 (s, 1H), 7.78 (d, J=8 Hz, 1H), 7.25 (d, J=8Hz, 1H), 6.56 (s, 1H), 6.31 (s, 1H), 5.85 (dd, J=10, 5 Hz, 1H), 5.35 (s,1H), 5.30 (d, J=10 Hz, 1H), 4.07 (t, J=14 Hz, 1H), 4.01 (m, 1H), 3.81(s, 3H), 3.76 (s, 3H), 3.65 (s, 3H), 3.14 (m, 1H), 2.82 (m), 2.83 (m,1H), 2.78 (s, 1H), 2.75 (s, 1H), 2.72 (s, 3H), 2.64 (s, 3H), 2.44 (m,2H), 2.31 (d, J=14 Hz, 1H), 2.08 (m, 1H), 2.01 (s, 3H), 1.88 (m, 1H),1.67 (m, 1H), 1.56-1.33 (m, 5H), 0.90 (t, J=7 Hz, 3H), 0.75 (t, J=7 Hz,3H); ESI m/z 853 [M+H]⁺.

Example 71 Preparation of 12′-(3-Methylbutanoyl)vincristine

A solution of 12′-(3-methylbutynyl)vincristine (75 mg, 0.08 mmol) informic acid (2 mL) was heated at 80° C. for 4 h. The reaction mixturewas cooled to room temperature and neutralized by the addition of solidsodium bicarbonate. Water was added and the mixture was extracted withmethylene chloride. The organic layer was dried (sodium sulfate),filtered, and concentrated under reduced pressure to give a brownresidue. Purification by flash chromatography (silica, 97:3 to 95:5CH₂Cl₂/MeOH) gave 12′-(3-methylbutanoyl)vincristine (27 mg, 35%) as atan solid: ¹H NMR (300 MHz, CD₃OD) δ 8.95 (s, 1H), 8.18 (s, 1H), 7.78(m, 2H), 7.32 (d, J=7 Hz, 1H), 7.23 (s, 1H), 6.88 (s, 1H), 5.89 (dd,J=10, 5 Hz, 1H), 5.40 (d, J=10 Hz, 1H), 5.19 (s, 1H), 4.57 (s, 1H),4.15-4.05 (m, 2H), 3.90 (s, 3H), 3.87 (m, 1H), 3.72 (s, 1H), 3.69 (s,3H), 3.62 (s, 3H), 3.40 (d, J=14 Hz, 1H), 3.32 (m), 3.17 (br d, J=14 Hz,1H), 3.06 (s, 1H), 3.00-2.75 (m, 5H), 2.62 (t, J=10 Hz, 1H), 2.47 (dd,J=14, 4 Hz, 1H), 2.33 (dd, J=14, 4 Hz, 1H), 2.25 (m, 1H), 2.08 (m, 1H),2.03 (s, 1H), 2.00 (s, 3H), 1.76 (m, 1H), 1.54-1.47 (m, 3H), 1.42-1.28(m, 2H), 1.34 (q, J=7 Hz, 2H), 1.01 (d, J=7 Hz, 6H), 0.90 (t, J=7 Hz,3H), 0.75 (t, J=7 Hz, 3H); ESI MS m/z 909 [M+H]⁺.

Example 72 Preparation of 12′-(Hexanoyl)vincristine

Preparation of 12′-(hexanoyl)vinblastine from 12′-(hexynyl)vinblastinewas carried out following the procedure described in Example 71 (29 mg,40%): ¹H NMR (300 MHz, CDCl₃) δ 8.95 (s, 1H), 8.18 (s, 1H), 7.78 (d, J=9Hz, 1H), 7.31 (d, J=9 Hz, 1H), 7.23 (s, 1H), 6.87(s, 1H), 5.89 (dd,J=10, 5 Hz, 1H), 5.41 (d, J=10 Hz, 1H), 5.17 (s, 1H), 4.58 (s, 1H), 4.12(d, J=14 Hz, 1H), 4.05 (m, 2H), 3.91 (s, 3H), 3.87 (s, 1H), 3.72 (s,1H), 3.69 (s, 3H), 3.63 (s, 3H), 3.42-3.00 (m, 7H), 2.83-2.70 (m, 3H),2.62 (br t, J=10 Hz, 1H), 2.43 (dd, J=15, 4 Hz, 1H), 2.29 (dd, J=15, 4Hz, 1H), 2.14-2.00 (m, 1H), 2.00 (s, 3H), 1.74 (m, 3H), 1.60-1.25 (m,11H), 0.93 (t, J =7 Hz, 3H), 0.90 (t, J=7 Hz, 3H), 0.73 (t, J=7 Hz, 3H);ESI MS m/z 923 [M+H]⁺.

Example 73 Preparation of 12′-(3-Methylbutyl)vincristine

To a solution of 12′-(3-methylbutynl)vincristine (15 mg, 0.02 mmol) intrifluoroacetic acid (1 mL) was added Et₃SiH (0.05 mg, 0.31 mmol) andthe reaction mixture was stirred overnight. Saturated aqueous sodiumbicarbonate was added to quench the reaction and the mixture wasextracted with methylene chloride. The organic layer was dried (sodiumsulfate), filtered, and concentrated under reduced pressure to give atan solid. Purification by flash column chromatography (silica, 95:5 to90:10 CH₂Cl₂/MeOH) gave 12′-(3-methylbutyl)vincristine (9 mg, 50%) as anoff-white solid: ¹H NMR (300 MHz, CD₃OD) δ 8.93 (s, 1H), 7.22 (s, 1H),7.15 (d, J=9 Hz, 1H), 6.96-6.94 (m, 2H), 5.90-5.88 (m, 1H), 5.40 (d,J=10 Hz, 1H), 5.15 (s, 1H), 4.58 (s, 1H), 4.09-4.07 (m, 1H), 3.90 (s,3H), 3.73-3.71 (m, 1H), 3.68 (s, 3H), 3.63 (s, 3H), 3.44-3.19 (m, 3H),3.07-3.05 (m, 2H), 2.89-2.65 (m, 6H), 2.49-2.39 (m, 2H), 2.03-2.01 (m,1H), 1.99 (s, 3H), 1.82-1.80 (m, 1H), 1.57-1.50 (m, 3H), 1.41-1.28 (m,9H), 0.96 (d, J=5 Hz, 6H), 0.89-0.78 (m, 8H); ESI MS m/z 895 [M+H]⁺.

Example 74 Preparation of 12′-Hexylvincristine

12′-hexylvincristine was prepared from 12′-(hexanoyl)vincristinefollowing the procedure described in Example 73 (87 mg, 44%). ¹H NMR(300 MHz, CD₃OD) δ 8.92 (s, 1H), 7.89 (s, 1H), 7.20 (s, 2H), 7.21 (d,J=8 Hz, 1H), 6.95 (s, 1H), 6.92 (d, J=8 Hz, 1H), 5.88 (dd, J=10, 5 Hz,1H), 5.40 (d, J=10 Hz, 1H), 5.15 (s, 1H), 4.58 (s, 1H), 4.08 (t, J=14Hz, 1H), 3.99-3.97 (m, 1H), 3.90 (s, 3H), 3.85 (s, 1H), 3.70 (s, 1H),3.67 (s, 3H), 3.63 (s, 3H), 3.45-3.15 (m, 2H), 3.08-3.06 (m, 2H),2.90-2.55 (m, 6H), 2.39 (dd, J=15, 5 Hz, 1H), 2.32 (dd, J=15, 5 Hz, 1H),2.04-2.03 (m, 1H), 2.00 (s, 3H), 1.81-1.79 (m, 1H), 1.60-1.20 (m, 16H),0.89 (t, J=7 Hz, 6H), 0.81 (t, J=7 Hz, 3H); ESI MS m/z 909 [M+H]⁺.

Example 75 Preparation of 12′-Methylvinblastine Trifluoroacetate

Dimethylzinc (2.0 M in toluene, 0.054 mL, 0.109 mmol) was added to12′-iodovinblastine (51 mg, 0.054 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (4.4 mg,0.005 mmol) in anhydrous 1,4-dioxane (2 mL) under nitrogen. The reactionmixture was heated at 45° C. for 45 min then quenched by the addition ofsaturated aqueous NaHCO₃ (3 mL). After extraction with chloroform (3×5mL), the combined organic extracts were washed with brine (5 mL), driedover MgSO₄, and evaporated to dryness in vacuo. The residue was purifiedby reverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′-methylvinblastine trifluoroacetate(9.4 mg, 16%): ¹H NMR (500 MHz, CD₃OD) δ 9.33 (s, 1H), 7.30 (s, 1H),7.16 (d, J=8.3 Hz, 1H), 6.97 (d, J=8.3 Hz, 1H), 6.68 (s, 1H), 6.42 (s,1H), 5.93 (dd, J=10.4, 4.1 Hz, 1H), 5.64 (d, J=10.5 Hz, 1H), 5.36 (s,1H), 4.61 (dd, J=16.8, 11.0, 1H), 3.92 (m, 3H), 3.86 (s, 3H), 3.82 (s,3H), 3.75-3.58 (m, 5H), 3.69 (s, 3H), 3.47 (d, J=15.7 Hz, 1H), 3.30 (m,1H), 3.21 (m, 1H), 2.88 (dd, J=14.3, 7.0 Hz, 1H), 2.77 (s, 3H),2.48-2.32 (m, 3H), 2.41 (s, 3H), 2.07 (s, 3H), 2.04 (m, 2H), 1.76 (m,1H), 1.66 (m, 2H), 1.52 (m, 3H), 1.38 (m, 1H), 0.97 (t, J=7.4 Hz, 3H),0.81 (t, J=7.3 Hz, 3H); ESI MS m/z 825 [M+H]⁺.

Example 76 Preparation of 12′-Methylvincristine Trifluoroacetate

Dimethylzinc (0.080 mL of a 2.0 M solution in toluene, 0.160 mmol) wasadded to 12′-iodovincristine (61 mg, 0.064 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium (II) (9.4 mg,0.012 mmol) in anhydrous 1,4-dioxane (1 mL) under nitrogen. The reactionmixture was heated to 80° C. for 2 h then quenched by the addition ofsaturated aqueous NaHCO₃ (3 mL). After extraction with EtOAc (2×5 mL),the combined organic extracts were dried (Na₂SO₄), and evaporated todryness in vacuo. The residue was purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid) toprovide 12′-methylvincristine trifluoroacetate (16 mg, 30%): ¹H NMR (500MHz, CD₃OD) δ 9.42 (br s, 1H), 8.98 (s, 1H), 7.33 (s, 1H), 7.30 (s, 1H),7.20 (d, J=8.5 Hz, 1H), 6.99-6.96 (m, 2H), 5.97 (dd, J=10.5, 5 Hz, 1H),7.73 (d. J=10.5 Hz, 1H), 5.18 (s, 1H), 4.67-4.64 (m, 2H), 4.06-4.02 (m,1H), 3.99-3.87 (m, 6H), 3.84-3.60 (m, 10H), 3.52 (d, J=15.5 Hz, 1H),3.34-3.12 (m, 1H), 3.16 (br s, 2H), 2.82 (dd, J=14.0, 6.0 Hz, 1H),2.51-2.48 (m, 1H), 2.41-2.37 (m, 4H), 2.07-1.96 (m, 4H), 1.67-1.57 (m,3H), 1.53-1.46 (m, 3H), 1.30-1.28 (m, 1H), 0.96 (t, J=7.0 Hz, 3H), 0.83(t, J=7.0 Hz, 3H); ESI MS m/z 839 [M+H]⁺.

Example 77 Preparation of 12′-Ethylvinblastine Trifluoroacetate

Diethylzinc (1.1 M in toluene, 0.103 mL, 0.113 mmol) was added to12′-iodovinblastine (53 mg, 0.057 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (4.6 mg,0.006 mmol) in anhydrous 1,4-dioxane (2 mL) under nitrogen. The reactionmixture was heated at 45° C. for 45 min then quenched by the addition ofsaturated aqueous NaHCO₃ (3 mL). After extraction with chloroform (3×4mL) the combined organic extracts were washed with brine (5 mL), driedover MgSO₄, and evaporated to dryness in vacuo. The residue was purifiedby reverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′-ethylvinblastine as a salt ofthrifluoroacetic acid (33.2 mg, 55%): ¹H NMR (500 MHz, CD₃OD) δ 9.34 (brs, 1H), 7.31 (s, 1H), 7.19 (d, J=8.3 Hz, 1H), 7.00 (dd, J=8.4, 1.1 Hz,1H), 6.68 (s, 1H), 6.42 (s, 1H), 5.94 (dd, J=10.5, 4.2 Hz, 1H), 5.64 (d,J=10.2 Hz, 1H), 5.36 (s, 1H), 4.84 (m, 1H), 4.62 (dd, J=17.0, 11.6, 1H),3.58-3.95 (m, 6H), 3.86 (s, 3H), 3.82 (s, 3H), 3.69 (s, 3H), 3.48 (d,J=15.8 Hz, 1H), 3.33 (m, 1H), 3.18 (m, 3H), 2.88 (dd, J=14.3, 6.0 Hz,1H), 2.77 (s, 3H), 2.71 (q, J=7.5 Hz, 2H), 2.46 (m, 1H), 2.36 (m, 1H),2.07 (s, 3H), 2.03 (m, 2H), 1.78 (m, 1H), 1.66 (m, 2H), 1.52 (m, 3H),1.39 (m, 1H), 1.24 (t, J=7.5 Hz, 3H), 0.97 (t, J =7.4 Hz, 3H), 0.81 (t,J=7.3 Hz, 3H); ESI MS m/z 839 [M+H]⁺.

Example 78 Preparation of 12′-Ethylvincristine Trifluoroacetate

Diethylzinc (0.174 mL of a 1.0 M solution in toluene, 0.174 mmol) wasadded to 12′-iodovincristine (66 mg, 0.069 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (10.2 mg,0.014 mmol) in anhydrous 1,4-dioxane (1 mL) under nitrogen. The reactionmixture was heated to 80° C. for 2 h then quenched by the addition ofsaturated aqueous NaHCO₃ (3 mL). After extraction with EtOAc (2×5 mL),the combined organic extracts were dried (Na₂SO₄), and evaporated todryness in vacuo. The residue was purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluroacetic acid) toprovide 12′-ethylvincristine trifluoroacetate (28 mg, 46%): ¹H NMR (500MHz, CD₃OD) δ 9.65 (br s, 1H), 8.98 (s, 1H), 7.33 (s, 2H), 7.31 (s, 1H),7.23 (d, J=8 Hz, 1H), 7.01-6.99 (m, 2H), 5.98 (dd, J=10.5, 5.0 Hz, 1H),5.73 (dd, J=10.5 Hz, 1H), 5.18 (s, 1H), 4.69-4.67 (m, 2H), 4.07-4.04 (m,1H), 4.00-3.87 (m, 6H), 3.84-3.58 (m, 8H), 3.54-3.51 (m, 2H), 3.36-3.35(m, 1H), 3.19-3.15 (m, 2H), 2.82 (dd, J=14.5, 6 Hz, 1H), 2.71 (q, J=10.0Hz, 2H), 2.51-2.49 (m, 1H), 2.41-2.36 (m, 1H), 2.08-1.94 (m, 5H),1.67-1.58 (m, 3H), 1.53-1.47 (m, 3H), 1.31-1.26 (m, 1H), 1.22 (t, J=8.0Hz, 3H), 0.96 (t, J=7.5 Hz, 3H), 0.83 (t, J=7.0 Hz, 3H); ESI MS m/z 853[M+H]⁺.

Example 79 Preparation of 12′-(N-Methyl-N-phenylamino)vinblastineTrifluoroacetate

12′-(N-Methyl-N-phenylamino)vinblastine was prepared according to thescheme below.

Step 1: A solution of 12′-iodovinblastine (308 mg, 0.328 mmol) in CH₂Cl₂(3 mL) was charged with N,N-diisopropylethylamine (575 μL, 3.28 mmol)and tert-butyldimethylsilyl trifluoromethanesulfonate (128 μL, 0.558mmol). After 2 h, the reaction mixture was diluted with saturatedaqueous NaHCO₃ (8 mL) and extracted with CH₂Cl₂ (2×10 mL). The combinedextracts were dried (Na₂SO₄) and concentrated to a brown solid which waspurified by flash chromatography (silica, [CHCl₃/MeOH/NH₄OH(40:18:2)]/CH₂Cl₂, 1:99 to 10:90) to yield12′-Iodo-3,4′-(tert-butyldimethylsilanyloxy)vinblastine (88 mg, 25%) asa white solid and 12′-Iodo-3-(tert-butyl-dimethylsilanyloxy)vinblastine(80 mg, 23%) as a white solid. The data for12′-Iodo-3,4′-(tert-butyl-dimethylsilanyloxy)vinblastine is as follows:ESI MS m/z 1165 [C₅₈H₈₅IN₄O₉Si₂+H]⁺.12′-Iodo-3-(tert-butyldimethylsilanyloxy) vinblastine: ESI MS m/z 1051[M+H]⁺.

Step 2: 12′-Iodo-3,4′-(tert-butyldimethylsilanyloxy)vinblastine (32 mg,0.030 mmol), N-methylaniline (7.4 mg, 0.070 mmol), and NaOt-Bu (9.2 mg,0.10 mmol) were dissolved in anhydrous toluene (1.0 mL) while stirringunder argon atmosphere in a sealed tube. The reaction mixture wasdeoxygenated with argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (2.5 mg, 2.7 μmol) and2-(dicyclohexyl-phosphino)-2′,4′,6′-tri-i-propyl-1, 1′-biphenyl (2.6 mg,10 μmol) were added. The reaction mixture was sealed and heated to 80°C. for 4 h. The reaction mixture was cooled to room temperature, dilutedwith EtOAc, filtered through diatomaceous earth, and then concentratedto provide crude12′-(N-methyl-N-phenylamino)-3,4′-(tert-butyl-dimethylsilanyloxy)vinblastine:ESI MS m/z 1144 [M+H]⁺.

Step 3: A solution of crude12′-(N-methylphenylamino)-3,4′-(tert-butyldimethylsilanyloxy)vinblastine(32 mg, 0.030 mmol) in THF (1.5 mL) was treated with tetrabutylammoniumfluoride (400 μL of a 1N solution in THF, 0.40 mmol). After 16 h, thereaction mixture was diluted with saturated aqueous NaHCO₃ (10 mL) andthen extracted with CH₂Cl₂ (2×10 mL). The combined organics were dried(Na₂SO₄), concentrated, and purified by purified by flash chromatography(silica gel, [CHCl₃/MeOH/NH₄OH (40:18:2)]/CH₂Cl₂, 1:99 to 10:90) toyield12′-(N-methyl-N-phenylamino)-4′-(tert-butyldimethylsilanyloxy)vinblastine:ESI MS m/z 1030 [M+H]⁺.

Step 4: A solution of12′-(N-methyl-N-phenylamino)-4′-(tert-butyldimethylsilanyloxy)vinblastine(32 mg, 0.030 mmol) was stirred HF•pyridine (100 μL) as a neat solutionin a flask. After 16 h, the reaction mixture was diluted with saturatedaqueous NaHCO₃ (10 mL) and then extracted with CH₂Cl₂ (2×10 mL). Thecombined organics were dried (Na₂SO₄), concentrated, and purified byreverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′-(N-methyl-N-phenylamino)vinblastinetrifluoroacetate (6 mg, 24%): ¹H NMR (500 MHz, CD₃OD) δ 9.67 (br s, 1H),7.31-7.28 (m, 2H), 7.11-7.09 (m, 2H), 6.94 (dd, J=9.0, 2.0 Hz, 1H), 6.76(s, 1H), 6.70-6.66 (m, 3H), 6.43 (s, 1H), 5.95 (dd, J=10.5, 4.5 Hz, 1H),5.66 (d, J=11.0 Hz, 1H), 5.36 (s, 1H), 4.61 (dd, J=17.0, 12.0 Hz, 1H),3.99-3.86 (m, 3H), 3.85-3.83 (m, 4H), 3.82-3.78 (m, 4H), 3.77-3.70 (m,5H), 3.65-3.51 (m, 3H), 3.34-3.22 (m, 6H), 3.16 (br s, 2H), 2.90 (dd,J=14.5, 4.5 Hz, 1H), 2.78 (s, 3H), 2.46-2.44 (m, 1H), 2.38-2.34 (m, 1H),2.07-2.02 (m, 4H), 1.78-1.74 (m, 1H), 1.65-1.64 (m, 1H), 1.60-1.49 (m,2H), 1.41-1.39 (m, 1H), 0.96 (t, J=7.5 Hz, 3H), 0.84 (t, J=7.5 Hz, 3H);ESI MS m/z 916 [M+H]⁺.

Example 80 Preparation of 12′-Aminovinblastine Trifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyldimethylsilanyloxy)-vinblastine (100 mg,0.0951 mmol), benzophenone imine (39 μL, 0.23 mmol), and NaOt-Bu (32 mg,0.33 mmol) were dissolved in anhydrous toluene (3 mL) while stirringunder argon atmosphere in a sealed tube. The mixture was deoxygenatedwith argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (8.7 mg, 9.5 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1, 1′-biphenyl (9.0 mg,19 μmol) were added. The reaction vessel was sealed and the mixtureheated to 80° C. for 3 h. The reaction mixture was cooled to roomtemperature, diluted with EtOAc, filtered through diatomaceous earth,and concentrated to provide crude12′-benzhydrylideneamino-3-(tert-butyldimethylsilanyloxy)-vinblastine.

Step 2: A solution of crude12′-benzhydrylideneamino-3-(tert-butyldimethylsilanyloxy)vinblastine(105 mg, 0.0952 mmol) in methanol (1.5 mL) was treated with NaOAc (54mg, 0.66 mmol) and hydroxylamine hydrochloride (33 mg, 0.47 mmol). After4 h, the reaction appeared complete as indicated by ESI mass spectralanalysis and the reaction mixture was concentrated to dryness. Theresidue was diluted with saturated aqueous NaHCO₃ (10 mL) and extractedwith CH₂Cl₂ (2×10 mL). The combined extracts were dried (Na₂SO₄), andthen concentrated to provide crude12′-amino-3-(tert-butyl-dimethylsilanyloxy)-vinblastine as a brown oil.A solution of crude12′-amino-3-(tert-butyldimethylsilanyloxy)vinblastine (89 mg, 0.094mmol) in THF (1.5 mL) was treated with tetrabutylammonium fluoride (450μL of a 1N solution in THF, 0.450 mmol). After 3 h, the reactionappeared complete as indicated by ESI mass spectral analysis. Thereaction mixture was diluted with saturated aqueous NaHCO₃ (10 mL) andextracted with EtOAc (2×10 mL). The combined extracts were dried(Na₂SO₄) and concentrated. The residue was purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid) toprovide 12′-aminovinblastine trifluoroacetate (32 mg, 32% yield over 3steps): ¹H NMR (500 MHz, CD₃OD) δ 10.24 (br s, 1H), 7.50 (d, J=2.0 Hz,1H), 7.43 (d, J=8.5 z, 1H), 7.11 (dd, J=8.5, 2.0 Hz, 1H), 6.70 (s, 1H),6.42 (s, 1H), 5.93 (dd, J=10.5, 4.0 Hz, 1H), 5.68 (d, J=10.5 Hz, 1H),5.34 (s, 1H), 4.74-4.72 (m, 1H), 3.98-3.90 (m, 4H), 3.86-3.84 (m, 4H),3.78-3.70 (m, 3H), 3.70-3.63 (m, 6H), 3.48 (d, J=16.0 Hz, 1H), 3.30-3.23(m, 2H), 3.19-3.17 (m, 2H), 2.89 (dd, J=14.5, 6.0 Hz, 1H), 2.79-2.77 (m,4H), 2.48-2.45 (m, 1H), 2.34-2.31 (m, 1H), 2.07-1.97 (m, 5H), 1.77-1.51(m, 6H), 1.41-1.35 (m, 1H), 0.97 (t, J=7.5 Hz, 3H), 0.78 (t, J=7.0 Hz,3H); ESI MS m/z 826 [M+H]⁺.

Example 81 Preparation of 12′-(N,N-Dimethylamino)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyl-dimethylsilanyloxy)vinblastine (31 mg,0.030 mmol), dimethylamine hydrochloride (6.0 mg, 0.070 mmol), andNaOt-Bu (17 mg, 0.18 mmol) were dissolved in anhydrous toluene (1.0 mL)while stirring under argon atmosphere in a sealed tube. The mixture wasdeoxygenated with an argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (2.7 mg, 2.9 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (2.8 mg,6.0 μmol) were added. The reaction vessel was sealed and the mixture washeated to 80° C. for 3.5 h. The reaction mixture was cooled to roomtemperature, diluted with EtOAc, filtered through diatomaceous earth,and then concentrated to provide crude12′-(N,N-dimethylamino)-3-(tert-butyldimethyl-silanyloxy)vinblastine.

Step 2: A solution of12′-(N,N-dimethylamino)-3-(tert-butyldimethylsilanyloxy)vinblastine(28.7 mg, 0.030 mmol) in THF (1.0 mL) was treated withtetrabutylammonium fluoride (400 μL of a 1 N solution in THF, 0.42mmol). After 3.5 h, the reaction mixture was diluted with saturatedaqueous NaHCO₃ (10 mL) and then extracted with CH₂Cl₂ (2×10 mL). Thecombined extracts were dried (Na₂SO₄), concentrated, and purified byreverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′-(N,N-dimethylamino)vinblastinetrifluoroacetate (8.8 mg, 25%): ¹H NMR (500 MHz, CD₃OD) δ 10.34 (br s,1H), 7.83 (d, J=2.0 Hz, 1H), 7.47 (d, J=8.5 Hz, 1H), 7.33 (dd, J=9.0,2.5 Hz, 1H), 6.69 (s, 1H), 6.42 (s, 1H), 5.95-5.92 (m, 1H), 5.68 (d,J=10.5 Hz, 1H), 5.34 (s, 1H), 4.75-4.71 (m, 1H), 3.97-3.81 (m, 10H),3.78-3.65 (m, 7H), 3.50-3.47 (m, 1H), 3.40-3.30 (m, 7H), 3.27-3.22 (m,1H), 3.19 (br s, 2H), 2.89 (dd, J=14.5, 6.5 Hz, 1H), 2.78 (s, 3H), 2.47(dd, J=16.0, 4.5 Hz, 1H), 2.35-2.32 (m, 1H), 2.07-1.99 (m, 4H),1.75-1.66 (m, 3H), 1.61-1.51 (m, 3H), 1.40-1.37 (m, 1H), 0.97 (t, J=7.5Hz, 3H), 0.77 (t, J=7.5 Hz, 3H); ESI MS m/z 854 [M+H]⁺.

Example 82 Preparation of 12′-(4-Methoxyphenylamino)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyldimethylsilanyloxy)vinblastine (33 mg,0.030 mmol), aniline (7.3 mg, 0.080 mmol), and NaOt-Bu (10 mg, 0.11mmol) were dissolved in anhydrous toluene (1.0 mL) while stirring underan argon atmosphere in a sealed tube. The mixture was deoxygenated withargon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (2.9 mg, 3.2 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (3.0 mg,6.4 μmol) were added. The reaction was sealed and heated to 80° C. for3.5 h. The reaction mixture was cooled to room temperature, diluted withEtOAc, filtered through diatomaceous earth, and then concentrated toprovide crude12′-(phenylamino)-3-(tert-butyldimethylsilanyloxy)vinblastine.

A solution of crude12′-(phenylamino)-3-(tert-butyldimethylsilanyloxy)vinblastine (32.7 mg,0.030 mmol) in THF (1.0 mL) was treated with tetrabutylammonium fluoride(400 μL of a 1 N solution in THF, 0.42 mmol). After 3.5 h, the reactionmixture was diluted with saturated aqueous NaHCO₃ (10 mL) and thenextracted with CH₂Cl₂ (2×10 mL). The combined organics were dried(Na₂SO₄), concentrated, and purified by reverse phase chromatography(C18, acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(phenylamino)vinblastine trifluoroacetate (6 mg, 20%): ¹H NMR (500MHz, CD₃OD) δ 7.21 (d, J=2.0 Hz, 1H), 7.13-7.09 (m, 3H), 6.95-6.90 (m,3H), 6.70-6.66 (m, 2H), 6.32 (s, 1H), 5.88-5.83 (m, 1H), 5.37 (s, 1H),5.29 (d, J=10.0 Hz, 1H), 4.08-3.95 (m, 2H), 3.83-3.81 (m, 4H), 3.76 (s,3H), 3.65 (s, 3H), 3.50-3.46 (m, 2H), 3.34-3.30 (m, 1H), 3.29-3.18 (m,3H), 2.99-2.95 (m, 1H), 2.82-2.78 (m, 3H), 2.71 (s, 3H), 2.51-2.47 (m,2H), 2.32-2.28 (m, 1H), 2.13-2.07 (m, 4H), 1.95-1.88 (m, 1H), 1.72-1.67(m, 1H), 1.54-1.49 (m, 1H), 1.44-1.39 (m, 2H), 1.35-1.28 (m, 3H), 0.90(t, J=7.5 Hz, 3H), 0.81 (t, J=7.5 Hz, 3H); ESI MS m/z 902 [M+H]⁺.

Example 83 Preparation of 12′-(4-Methoxyphenylamino)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyl-dimethylsilanyloxy)vinblastine (54.4 mg,0.0501 mmol), p-anisidine (14 mg, 0.13 mmol), and NaOt-Bu (17 mg, 0.18mmol) were dissolved in anhydrous toluene (1.0 mL) while stirring underargon atmosphere in a sealed tube. The reaction mixture was deoxygenatedwith argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (4.7 mg, 5.2 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (4.9 mg,10 μmol) were added. The reaction vessel was sealed and heated to 80° C.for 4 h. The reaction mixture was cooled to room temperature, dilutedwith EtOAc, filtered through diatomaceous earth, and then concentratedto provide crude12′-(4-methoxyphenylamino)-3-(tert-butyl-dimethylsilanyloxy)vinblastine:ESI MS m/z 1046 [M+H]⁺.

Step 2: A solution of12′-(4-methoxyphenylamino)-3-(tert-butyldimethylsilanyloxy)vinblastine(54 mg, 0.051 mmol) in THF (1.0 mL) was treated with tetrabutylammoniumfluoride (154 μL of a 1 N solution in THF, 0.154 mmol). After 4 h, thereaction mixture appeared complete as indicated by ESI mass spectralanalysis. The reaction mixture was diluted with saturated aqueous NaHCO₃(10 mL) and extracted with CH₂Cl₂ (2×10 mL). The combined extracts weredried (Na₂SO₄), concentrated, and purified by reverse phasechromatography (C18, acetonitrile/water, 0.05% trifluoroacetic acid) toprovide 12′-(4-methoxyphenylamino)vinblastine trifluoroacetate (23 mg,48%): ¹H NMR (500 MHz, CD₃OD) δ 9.98 (br s, 1H), 7.33 (s, 1H), 7.27 (d,J=8.5 Hz, 1H), 7.03 (d, J=7 Hz, 2H), 6.91 (d, J=8.0 Hz, 1H), 6.82 (d,J=8.5 Hz, 2H), 6.63 (s, 1H), 6.42 (s, 1H), 5.94 (dd, J=10.5, 4.0 Hz,1H), 5.85 (d, J=4.5 Hz, 1H), 5.63 (d, J=9.5 Hz, 1H), 5.32 (s, 1H), 4.76(d, J=14.5 Hz, 1H), 4.65-4.63 (m, 1H), 4.03 (d, J=16.5 Hz, 1H), 3.94(dd, J=15.0, 5.0 Hz, 1H), 3.92-3.87 (m, 4H), 3.85 (m, 4H), 3.78-3.71 (m,10H), 3.47-3.44 (m, 1H), 3.45-3.29 (m, 2H), 3.20-3.08 (m, 2H), 2.82 (dd,J=13.5, 4.5 Hz, 1H), 2.78 (s, 3H), 2.63-2.57 (m, 1H), 2.37-2.32 (m, 1H),2.17-2.03 (m, 7H), 1.95-1.92 (m, 1H), 1.70-1.64 (m, 1H), 1.12 (t, J=7.5Hz, 3H), 0.76 (t, J=7.0 Hz, 3H); ESI MS m/z 932 [C₅₃H₆₅N₅O₁₀+H]⁺.

Example 84 Preparation of 12′-(4-Trifluoromethylphenylamino)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyldimethylsilanyloxy)vinblastine (31 mg,0.030 mmol), 4-trifluoromethylaniline (11 mg, 0.070 mmol), and NaOt-Bu(9.0 mg, 0.090 mmol) were dissolved in anhydrous toluene (1.0 mL) whilestirring under an argon atmosphere in a sealed tube. The mixture wasdeoxygenated with argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (2.4 mg, 2.6 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (2.5 mg,5.3 μmol) were added. The reaction was sealed and heated to 80° C. for 3h. The reaction mixture was cooled to room temperature, diluted withEtOAc, filtered through diatomaceous earth, and then concentrated toprovide crude12′-(4-trifluoromethylphenylamino)-3-(tert-butyldimethylsilanyloxy)vinblastine.

Step 2: A solution of crude12′-(4-trifluoromethylphenylamino)-3-(tert-butyldimethylsilanyloxy)vinblastine(29 mg, 0.030 mmol) in THF (1.0 mL) was treated with tetrabutylammoniumfluoride (700 μL of a 1 N solution in THF, 0.70 mmol). After 16 h, thereaction mixture was diluted with saturated aqueous NaHCO₃ (10 mL) andthen extracted with CH₂Cl₂ (2×10 mL). The combined organics were dried(Na₂SO₄), concentrated, and purified by reverse phase chromatography(C18, acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(4-trifluoromethylphenylamino)vinblastine trifluoroacetate (5 mg,19%): ¹H NMR (500 MHz, CD₃OD) δ 7.37-7.33 (m, 3H), 7.26 (d, J=8.5 Hz,1H), 7.02 (dd, J=8.5, 1.5 Hz, 1H), 6.94 (d, J=8.5 Hz, 2H), 6.58 (br s,1H), 6.39 (s, 1H), 5.90 (dd, J=9.5, 4.5 Hz, 1H), 5.47-5.43 (m, 1H), 5.35(s, 1H), 4.61-4.58 (m, 1H), 3.96-3.90 (m, 2H), 3.85 (s, 3H), 3.81-3.79(m, 4H), 3.69-3.57 (m, 8H), 3.44-3.24 (m, 4H), 3.17 (br s, 2H),2.89-2.88 (m, 1H), 2.74 (s, 3H), 2.49-2.46 (m, 1H), 2.23-2.21 (m, 1H),2.05 (s, 3H), 1.92-1.90 (m, 1H), 1.74-1.66 (m, 2H), 1.53-1.38 (m, 3H),1.29-1.27 (m, 1H), 0.96 (t, J=7.5 Hz, 3H), 0.80 (t, J=7.0 Hz, 3H); ESIMS m/z 970 [M+H]⁺.

Example 85 Preparation of 12′-(4-Piperidinyl)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyldimethylsilanyloxy)vinblastine (23 mg,0.022 mmol), piperidine (5.4 mg, 0.055 mmol), and NaOt-Bu (7.4 mg, 0.077mmol) were dissolved in anhydrous toluene (1.0 mL) while stirring underargon atmosphere in a sealed tube. The mixture was deoxygenated withargon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (2.0 mg, 2.2 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (2.1 mg,4.4 μmol) were added. The reaction vessel was sealed and heated to 80°C. for 4 h. The reaction mixture was cooled to room temperature, dilutedwith EtOAc, filtered through diatomaceous earth, and then concentratedto provide crude12′-(1-piperidinyl)-3-(tert-butyldimethylsilanyloxy)vinblastine: ESI MSm/z 1008 [M+H]⁺.

Step 2: A solution of12′-(1-piperidinyl)-3-(tert-butyldimethylsilanyloxy)vinblastine (24 mg,0.023 mmol) in THF (1.0 mL) was treated with tetrabutylammonium fluoride(95 μL of a 1 N solution in THF, 0.095 mmol). After 3 h, the reactionappeared complete as indicated by ESI mass spectral analysis. Thereaction mixture was diluted with saturated aqueous NaHCO₃ (10 mL) andextracted with CH₂Cl₂ (2×10 mL). The combined extracts were dried(Na₂SO₄), concentrated, and purified by reverse phase chromatography(C18, acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(1-piperidinyl)vinblastine trifluoroacetate (6.1 mg, 25%): ¹H NMR(500 MHz, CD₃OD) δ 10.36 (br s, 1H), 7.84 (d, J=2.0 Hz, 1H), 7.47 (d,J=8.5 Hz, 1H), 7.35 (dd, J=8.5, 2.0 Hz, 1H), 6.69 (s, 1H), 6.40 (s, 1H),5.95-5.92 (m, 1H), 5.68 (d, J=10.5 Hz, 1H), 5.34 (s, 1H), 4.77-4.71 (m,1H), 3.98-3.81 (m, 9H), 3.77-3.62 (m, 7H), 3.50-3.34 (m, 3H), 3.33-3.19(m, 3H), 2.89 (dd, J=14.5, 6.0 Hz, 1H), 2.77-2.76 (m, 3H), 2.50-2.46 (m,1H), 2.37-2.31 (m, 1H), 2.07-1.98 (m, 8H), 1.77-1.35 (m, 13H), 0.97 (t,J=7.5 Hz, 3H), 0.77 (t, J=7.5 Hz, 3H); ESI MS m/z 894 [M+H]⁺.

Example 86 Preparation of 12′-(4-Morpholino)vinblastine Trifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyldimethylsilanyloxy)vinblastine (23.3 mg,0.022 mmol), piperidine (6.8 mg, 0.055 mmol), and NaOt-Bu (7.4 mg, 0.077mmol) were dissolved in anhydrous toluene (1.0 mL) while stirring underargon atmosphere in a sealed tube. The reaction mixture was deoxygenatedwith argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (2.0 mg, 2.2 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (2.1 mg,4.4 μmol) were added. The reaction mixture was sealed and heated to 80°C. for 4 h. The reaction mixture was cooled to room temperature, dilutedwith EtOAc, filtered through diatomaceous earth, and then concentratedto provide crude12′-(4-morpholino)-3-(tert-butyldimethylsilanyloxy)vinblastine: ESI MSm/z 1010 [M+H]⁺.

Step 2: A solution of12′-(4-morpholino)-3-(tert-butyldimethylsilanyloxy)vinblastine (24 mg,0.023 mmol) in THF (1.0 mL) was treated with tetrabutylammonium fluoride(95 μL in a 1 N solution in THF, 0.095 mmol). After 3 h, the reactionappeared complete as indicated by ESI mass spectral analysis. Thereaction mixture was diluted with saturated aqueous NaHCO₃ (10 mL) andextracted with CH₂Cl₂ (2×10 mL). The combined extracts were dried(Na₂SO₄), concentrated, and purified by reverse phase chromatography(C18, acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(4-morpholino)vinblastine trifluoroacetate (5.1 mg, 20: ¹H NMR (500MHz, CD₃OD) δ 9.91 (br s, 1H), 7.48 (s, 1H), 7.36 (d, J=9.0 Hz, 1H),7.18 (d, J=7.0 Hz, 1H), 6.88 (s, 1H), 6.42 (s, 1H), 5.93 (dd, J=10.5,4.5 Hz, 1H), 5.66 (d, J=11.0 Hz, 1H), 5.35 (s, 1H), 4.71-4.65 (m, 1H),4.02-3.89 (m, 5H), 3.86 (s, 3H), 3.81 (s, 3H), 3.77-3.71 (m, 2H),3.69-3.63 (m, 5H), 3.49-3.40 (m, 5H), 3.36-3.18 (m, 6H), 2.88-2.87 (m,1H), 2.78 (s, 3H), 2.47 (dd, J=16.5, 5.5 Hz, 1H), 2.38-2.32 (m, 1H),2.07-1.98 (m, 5H), 1.77-1.63 (m, 4H), 1.58-1.51 (m, 2H), 1.43-1.37 (m,1H), 0.97 (t, J=7.5 Hz, 3H), 0.79 (t, J=7.5 Hz, 3H); ESI MS m/z 896[M+H]⁺.

Example 87 Preparation of 12′-(Pyrrolidin-1-yl)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyldimethylsilanyloxy)vinblastine (100 mg,0.10 mmol), pyrrolidine (19 μL, 0.24 mmol), and NaOt-Bu (32 mg, 0.33mmol) were dissolved in anhydrous toluene (2.0 mL) while stirring underan argon atmosphere in a sealed tube. The reaction mixture wasdeoxygenated with argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (8.7 mg, 9.5 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (9.1 mg,20 μmol) were added. The reaction mixture was sealed and heated to 80°C. for 2 h. The reaction mixture was cooled to room temperature, dilutedwith EtOAc, filtered through diatomaceous earth, and then concentrated.Purification of the residue by flash chromatography (silica, 8:1 to 1:1EtOAc/hexanes) gave12′-(pyrrolidin-1-yl)-3-(tert-butyldimethylsilanyloxy)vinblastine: ESIMS m/z 994 [M+H]⁺.

Step 2: A solution of12′-(pyrrolidin-1-yl)-3-(tert-butyldimethylsilanyloxy)vinblastine (12mg, 0.012 mmol) in THF (2.0 mL) was treated with tetrabutylammoniumfluoride (1 mL of a 1 N solution in THF). After 3 h, the reactionappeared complete as indicated by ESI mass spectral analysis. Thereaction mixture was diluted with saturated aqueous NaHCO₃ (10 mL) andthen extracted with EtOAc (2×10 mL). The combined extracts were dried(Na₂SO₄) and concentrated. Purification by reverse phase chromatography(C18, acetonitrile/water, 0.05% trifluoroacetic acid) gave12′-(pyrrolidin-1-yl)vinblastine trifluoroacetate (2 mg, 2%): ESI MS m/z880 [M+H]⁺.

Example 88 Preparation of 12′-(Azetidin-1-yl)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyldimethylsilanyloxy)vinblastine (716 mg,0.68 mmol), azetidine (160 μL, 2.3 mmol), and NaOt-Bu (229 mg, 2.38mmol) were dissolved in anhydrous toluene (5.0 mL) while stirring underargon atmosphere in a sealed tube. The reaction mixture was deoxygenatedwith argon at room temperature for 3 min thentris(dibenzylideneacetone)dipalladium(0) (62 mg, 68 μmol) and2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (64 mg,140 μmol) were added. The reaction mixture was sealed and heated to 60°C. for 3 h. The reaction mixture was cooled to room temperature, dilutedwith EtOAc, filtered through diatomaceous earth, and then concentrated:ESI MS m/z 980 [M+H]⁺.

Step 2: A solution of crude12′-(azetidin-1-yl)-3-(tert-butyldimethylsilanyloxy)vinblastine in THF(2.0 mL) was treated with tetrabutylammonium fluoride (1 mL in a 1 Nsolution in THF). After 3 h, the reaction appeared complete as indicatedby ESI mass spectral analysis. The reaction mixture was diluted withsaturated aqueous NaHCO₃ (10 mL) and extracted with EtOAc (2×10 mL). Thecombined extracts were dried (Na₂SO₄), concentrated, and purified byreverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′-(azetidin-1-yl)vinblastine (230 mg,39%) as a trifluoroacetic acid salt which was converted to an L-tartaricacid salt (200 mg, 64% yield). 12′-(Azetidin-1-yl)vinblastine L-tartaricacid was isolated as a white powder: ¹H NMR (500 MHz, CD₃OD) 7.18-7.13(m, 1H), 6.62 (s, 1H), 6.54-6.51 (m, 2H), 6.35 (s, 1H), 5.88-5.85 (m,1H), 5.38-5.32 (m, 2H), 4.58-4.50 (m, 1H), 4.42 (s, 4H), 3.90-3.82 (m,6H), 3.79-3.75 (m, 3H), 3.69-3.60 (m, 6H), 3.49-3.40 (m, 2H), 3.35-3.27(m, 4H), 3.26-3.15 (m, 3H), 2.95-2.83 (m, 3H), 2.77-2.70 (m, 4H),2.46-2.34 (m, 2H), 2.16-2.12 (m, 1H), 2.07-2.05 (m, 3H), 1.93-1.88 (m,1H), 1.70-1.61 (m, 3H), 1.53-1.48 (m, 2H), 1.47-1.32 (m, 2H), 0.96 (t,J=7.5 Hz, 3H), 0.76 (t, J=7.5 Hz, 3H); ESI MS m/z 866 [M+H]⁺.

Example 89 Preparation of 12′-(3-Methylpyrazol-1-yl)vinblastineTrifluoroacetate

Step 1: 12′-Iodo-3-(tert-butyl-dimethylsilanyloxy)vinblastine (34 mg,0.030 mmol), 3-methyl-1H-pyrazole (5.9 μL, 0.070 mmol), and K₂CO₃ (14mg, 0.10 mmol) were dissolved in anhydrous toluene (1.0 mL) whilestirring under argon atmosphere in a sealed tube. The mixture wasdeoxygenated with argon at room temperature for 3 min then copper (I)iodide (2.7 mg, 2.9 μmol) and N,N-dimethylethylenediamine (5.6 μL, 10μmol) were added. The reaction vessel was sealed and the mixture washeated to 80° C. for 2 days. The reaction mixture was cooled to roomtemperature, diluted with EtOAc, filtered through diatomaceous earth,and then concentrated to provide crude12′-(3-methylpyrazol-1-yl)-3-(tert-butyldimethylsilanyloxy)vinblastine:ESI MS m/z 1005 [M+H]⁺.

Step 2: A solution of crude12′-(3-methylpyrazol-1-yl)-3-(tert-butyldimethylsilanyloxy)vinblastine(30 mg, 0.030 mmol) in THF (1.0 mL) was treated with tetrabutylammoniumfluoride (1.2 mL of a 1 N solution in THF, 1.2 mmol). After 5 h, thereaction mixture was diluted with saturated aqueous NaHCO₃ (10 mL) andthen extracted with CH₂Cl₂ (2×10 mL). The combined organics were dried(Na₂SO₄), concentrated, and purified by reverse phase chromatography(C18, acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′-(3-methylpyrazol-1-yl)vinblastine trifluoroacetate (5.0 mg, 19%): ¹HNMR (500 MHz, CD₃OD) δ 9.92 (br s, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.77 (d,J=2.0 Hz, 1H), 7.44-7.41 (m, 1H), 7.37 (d, J=8.5 Hz, 1H), 6.70 (s, 1H),6.44 (d, J=2.5 Hz, 1H), 6.29 (d, J=2.0 Hz, 1H), 5.94 (dd, J=10.5, 4.0Hz, 1H), 5.68 (d, J=10.5 Hz, 1H), 5.36 (d, J=3.0 Hz, 1H), 4.71-4.67 (m,1H), 3.99-3.90 (m, 3H), 3.87-3.84 (m, 4H), 3.82 (s, 3H), 3.78-3.73 (m,3H), 3.70-3.65 (m, 4H), 3.51-3.48 (m, 1H), 3.38-3.31 (m, 2H), 3.27-3.16(m, 3H), 2.91 (dd, J=14.0, 6.0 Hz, 1H), 2.78 (s, 3H), 2.48 (dd, J=15.5,4.5 Hz, 1H), 2.38-2.34 (m, 4H), 2.08-2.02 (m, 5H), 1.78-1.74 (m, 1H),1.67-1.66 (m, 1H), 1.60-1.50 (m, 2H), 1.41-1.38 (m, 1H), 0.97 (t, J=7.0Hz, 3H), 0.82 (t, J=7.5 Hz, 3H); ESI MS m/z 891 [M+H]⁺.

Example 90 Preparation of 12′,13′-Diiodovincristine Trifluoroacetate

An ice cold solution of N-iodosuccinimide (65 mg, 0.288 mmol) in 1:1trifluoroacetic acid/CH₂Cl₂ (5.0 mL) was added dropwise over 30 minutesto a solution of vincristine sulfate (188 mg, 0.190 mmol) in 1:1trifluoroacetic acid/CH₂Cl₂ (5.0 mL) at 0° C. When the reaction wascomplete as indicated by HPLC, the reaction mixture was poured into asolution of saturated aqueous NaHCO₃ (50 mL) and 10% aqueous NaHSO₃ (30mL) and then extracted with EtOAc (2×30 mL). The combined organics werewashed with brine (30 mL), dried over Na₂SO₄, and evaporated to drynessin vacuo. The residue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′,13′-diiodovincristine trifluoroacetate (44 mg, 18%): ¹H NMR (500MHz, CD₃OD) δ 10.24 (br s, 1H), 8.98 (s, 1H), 8.14 (s, 1H), 7.99 (s,1H), 7.34 (s, 1H), 6.88-6.64 (m, 1H), 6.00-5.97 (m, 1H), 5.74 (d, J=10Hz, 1H), 5.18 (s, 1H), 4.67-4.58 (m, 2H), 4.01-3.90 (m, 5H), 3.82-3.66(m, 8H), 3.59-3.43 (m, 3H), 3.35-3.25 (m, 1H), 3.19-3.15 (m, 3H),2.88-2.82 (m, 1H), 2.53-2.48 (m, 1H), 2.41-2.38 (m, 1H), 2.04-2.02 (m,4H), 1.95-1.93 (m, 1H), 1.66-1.63 (m, 3H), 1.55-1.50 (m, 3H), 1.32-1.27(m, 1H), 0.97-0.95 (m, 3H), 0.82-0.79 (m, 3H); ESI MS m/z 1077 [M+H]⁺.

Example 91 Preparation of 12′,13′-Diiodovinblastine Trifluoroacetate

An ice cold solution of N-iodosuccinimide (112 mg, 0.498 mmol) in 1:1trifluoroacetic acid/CH₂Cl₂ (10 mL) was added dropwise over 40 minutesto a solution of vinblastine sulfate (261 mg, 0.249 mmol) in 1:1trifluoroacetic acid/CH₂Cl₂ (10 mL) at 0° C. When the reaction wascomplete as indicated by HPLC, the reaction mixture was poured into asolution of saturated aqueous NaHCO₃ (50 mL) and 10% aqueous NaHPO₃ (30mL) and then extracted with EtOAc (2×30 mL). The combined organics werewashed with brine (30 mL), dried over Na₂SO₄, and evaporated to drynessin vacuo. The residue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluoroacetic acid) to provide12′,13′-diiodovinblastine trifluoroacetate (79.5 mg, 25%): ¹H NMR (500MHz, DMSO-d₆) 10.34 (br s, 1H), 8.17 (s, 1H), 7.97 (s, 1H), 6.70 (s,1H), 6.45 (s, 1H), 5.88 (dd, J=11.0, 5.5 Hz, 1H), 5.68 (d, J=10.5 Hz,1H), 5.15-5.13 (m, 2H), 4.33-4.27 (m, 1H), 3.95-3.91 (m, 1H), 3.88-3.77(m, 7H), 3.73-3.60 (m, 8H), 3.55-3.42 (m, 3H), 3.23-3.05 (m, 3H),2.83-2.81 (m, 1H), 2.70 (s, 3H), 2.30-2.28 (m, 1H), 2.17-2.15 (m, 1H),2.03 (s, 3H), 1.83-1.79 (m, 1H), 1.58-1.39 (m, 5H), 1.15-1.12 (m, 1H),0.85 (t, J=7.0 Hz, 3H), 0.63 (t, J=7.0 Hz, 3H); ESI MS m/z 1063 [M+H]⁺.

Example 92 Preparation of 13′-Iodo-12′-methylvincristineTrifluoroacetate

A ice cold solution of N-iodosuccinimide (8.2 mg, 0.037 mmol) in 1:1trifluoroacetic acid/CH₂Cl₂ (4.0 mL) was added dropwise over 30 minutesto a solution of 12′-methylvincristine (31 mg, 0.37 mmol) in 1:1trifluoroacetic acid/CH₂Cl₂ (4.0 mL) at 0° C. When the reaction wascomplete as indicated by HPLC, the reaction mixture was poured into asolution of saturated aqueous NaHCO₃ (50 mL) and 10% aqueous NaHSO₃ (30mL) and the resulting mixture was extracted with EtOAc (2×30 mL). Thecombined organics were washed with brine (30 mL), dried (Na₂SO₄), andevaporated to dryness in vacuo. The residue was purified by reversephase chromatography (C18, acetonitrile/water, 0.05% trifluoroaceticacid) to provide 13′-iodo-12′-methylvincristine trifluoroacetate (6 mg,17%): ¹H NMR (500 MHz, DMSO-d₆) δ 10.18 (br s, 1H), 9.07 (s, 1H), 7.87(s, 1H), 7.53 (s, 1H), 7.43 (s, 1H), 6.98 (s, 1H), 5.88 (dd, J=10.0, 4.5Hz, 1H), 5.58-5.54 (m, 1H), 5.14 (brs, 1H), 5.00 (s, 1H), 4.56 (br s,1H), 4.39-4.33 (m, 1H), 3.92-3.81 (m, 5H), 3.72-3.45 (m, 10H), 3.21-3.18(m, 1H), 3.13-3.05 (m, 2H), 2.71-2.68 (m, 1H), 2.51-2.45 (m, 4H),3.99-3.97 (m, 1H), 2.23-2.18 (m, 2H), 2.00 (s, 3H), 1.77-1.75 (m, 1H),1.56-1.52 (m, 1H), 1.46-1.40 (m, 5H), 1.08-1.06 (m, 1H), 0.86 (t, J=7.5Hz, 3H), 0.67 (t, J=7.0 Hz, 3H); ESI MS m/z 965 [M+H]⁺.

Example 93 Preparation of 12′,13′-Dimethylvincristine Trifluoroacetate

Dimethylzinc (0.162 mL of 2.0 M solution in toluene, 0.324 mmol) wasadded to 12′,13′-diiodovincristine (70 mg, 0.064 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (9.4 mg,0.013 mmol) in anhydrous 1,4-dioxane under nitrogen. The reactionmixture was heated to 80° C. for 3 h then quenched by the addition ofsaturated aqueous NaHCO₃ (10 mL). After extraction with CH₂Cl₂ (2×10mL), the combined organics were washed with brine (5 mL), dried(Na₂SO₄), and evaporated to dryness in vacuo. The residue was purifiedby reverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′,13′-dimethylvincristinetrifluoroacetate (12 mg, 21%): ¹H NMR (500 MHz, CD₃OD) δ 9.46 (br s,1H), 8.98 (s, 1H), 7.33 (s, 1H), 7.25 (s, 1H), 7.11 (s, 1H), 6.95 (d,J=3.0 Hz, 1H), 5.96 (dd, J=10.0, 4.5 Hz, 1H), 5.71 (d, J=10.5, 1H), 5.18(s, 1H), 4.67 (s, 1H), 4.56-4.48 (m, 1H), 3.95-3.87 (m, 7H), 3.79-3.66(m, 9H), 3.61-3.56 (m, 1H), 3.48-3.44 (m, 1H), 3.34-3.27 (m, 1H),3.24-3.15 (m, 2H), 2.83 (dd, J=14.5, 6.5 Hz, 1H), 2.54-2.48 (m, 1H),2.41-2.29 (m, 7H), 2.08-1.93 (m, 4H), 1.67-1.59 (m, 3H), 1.52-1.45(m,3H), 1.33-1.29 (m, 1H), 0.96 (t, J=7.5 Hz, 3H), 0.84 (t, J=7.5 Hz, 3H);ESI MS m/z 853 [C₄₈H₆₀N₄O₁₀+H]⁺.

Example 94 Preparation of 13′-Ethyl-12′-methylvincristineTrifluoroacetate

Diethylzinc (0.074 mL of a 1.0 M solution in toluene, 0.074 mmol) wasadded to 13′-iodo-12′-methylvincristine (28 mg, 0.029 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (21 mg,0.029 mmol) in anhydrous 1,4-dioxane (1 mL) under nitrogen. The reactionmixture was heated to 80° C. for 2 h then quenched by the addition ofsaturated aqueous NaHCO₃ (3 mL). After extraction with EtOAc (2×5 mL),the combined organics were dried (Na₂SO₄), and evaporated to dryness invacuo. The residue was purified by reverse phase chromatography (C18,acetonitrile/water, 0.05% trifluoroacetic acid) to provide13′-ethyl-12′-methylvincristine trifluoroacetate (5.0 mg, 19%): ¹H NMR(500 MHz, CD₃OD) δ9.53 (br s, 1H), 8.92 (s, 1H), 7.32 (s, 1H), 7.25 (s,1H), 7.13 (s, 1H), 6.94 (s, 1H), 5.96 (dd, J=10.4, 5.6 Hz, 1H), 5.70 (d,J=10.4 Hz, 1H), 5.18 (s, 1H), 4.66 (s, 1H), 4.61 (dd, J=18.0, 11.5 Hz,1H), 3.98-3.87 (m, 6H), 3.75-3.72 (m, 4H), 3.68-3.57 (m, 5H), 3.44-3.27(m, 3H), 3.20-3.13 (m, 3H), 2.82 (dd, J=7.0, 14.5 Hz, 1H), 2.70-2.64 (m,2H), 2.51-2.47 (m, 1H), 2.39-2.35 (m, 4H), 2.06-1.91 (m, 4H), 1.67-1.59(m, 3H), 1.53-1.48 (m, 3H), 1.31-1.28 (m, 1H), 1.18 (t, J=7.5 Hz, 3H),0.96 (t, J=7.5 Hz, 3H), 0.85 (t, J=7.5 Hz, 3H); ESI MS m/z 867 [M+H]⁺.

Example 95 Preparation of 12′,13′-Diethylvincristine Trifluoroacetate

Diethylzinc (0.308 mL of a 1.0 M solution in toluene, 0.308 mmol) wasadded to 12′,13′-diiodovincristine (66 mg, 0.061 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (13.5 mg,0.018 mmol) in anhydrous 1,4-dioxane under nitrogen. The reactionmixture was heated to 80° C. for 30 minutes then quenched by theaddition of saturated aqueous NaHCO₃ (10 mL). After extraction withCH₂Cl₂ (2×10 mL), the combined organics were washed with brine (5 mL),dried (Na₂SO₄), and evaporated to dryness in vacuo. The residue waspurified by reverse phase chromatography (C18, acetonitrile/water, 0.05%trifluoroacetic acid) to provide 12′,13′-diethylvincristinetrifluoroacetate (25 mg, 36%): ¹H NMR (500 MHz, DMSO-d₆) δ 11.13 (br s,1H), 9.08 (s, 1H), 7.43 (s, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 7.11 (s,1H), 5.88 (dd, J=10.5, 5.5 Hz, 1H), 5.59 (d, J=10.5 Hz, 1H), 5.19-5.12(m, 1H), 5.01 (s, 1H), 4.59 (s, 1H), 4.40-4.35 (m, 1H), 4.05-4.01 (m,1H), 3.93-3.78 (m, 6H), 3.72-3.62 (m, 5H), 3.56-3.53 (m, 4H), 3.41-3.37(m, 1H), 3.23-2.18 (m, 1H), 3.12-3.10 (m, 2H), 3.05-3.01 (m, 1H),2.77-2.57 (m, 4H), 2.29-2.17 (m, 2H), 2.03-2.00 (m, 4H), 1.87-1.82 (m,1H), 1.56-1.39 (m, 6H), 1.19 (t, J=7.5 Hz, 3H), 1.12 (t, J=7.5 Hz, 3H),0.70 (t, J=7.5 Hz, 3H), 0.65 (t, J=7.0 Hz, 3H); ESI MS m/z 881 [M+H]⁺.

Example 96 Preparation of 13′-Acetyl-12′-diethylvincristineTrifluoroacetate

Step 1: 13′-Iodo-12′-methylvincristine (28 mg, 0.030 mmol), copper(I)iodide (0.83 mg, 0.0044 mmol), dichlorobis(triphenylphosphine)palladium(II) (2.0 mg, 0.0029 mmol), toluene (2 mL), and triethylamine (1 mL)were combined in a resealable glass test tube. Argon was bubbled throughthe solution for 3 min, then (trimethylsilyl)acetylene (24 μL, 0.17mmol) was added and the mixture was heated at 55° C. for 1 h. Saturatedaqueous NaHCO₃ (5 mL) was added and the mixture was extracted with ethylacetate (3×5 mL). The combined organics were washed with brine (5 mL),dried over Na₂SO₄, and evaporated to dryness in vacuo to yield crude12′-methyl-13′-trimethylsilanylethynylvincristine: ESI MS m/z 935 [M+H]⁺

Step 2: Crude 12′-methyl-13′-trimethylsilanylethynylvincristine (27 mg,0.028 mmol) was added to a solution of CH₂Cl₂ (1 mL) and trifluoroaceticacid (1 mL). The reaction was monitored by ESI MS. After 30 min, thereaction mixture was poured into a solution of saturated aqueous NaHCO₃(50 mL) and the resulting mixture was extracted with EtOAc (2×30 mL).The combined organics were washed with brine (30 mL), dried (Na₂SO₄),and evaporated to dryness in vacuo. The residue was purified by reversephase chromatography (C18, acetonitrile/water, 0.05% trifluoroaceticacid) to provide 13′-acetyl-12′-methylvincristine trifluoroacetate (6mg, 17%): ¹H NMR (500 MHz, CD₃OD) δ 10.24 (br s, 1H), 8.99 (s, 1H), 7.90(s, 1H), 7.39 (s, 1H), 7.35 (s, 1H), 6.95 (s, 1H), 5.97 (dd, J=10.0, 4.5Hz, 1H), 5.73 (d, J=10.0 Hz, 1H), 5.19 (s, 1H), 4.68-4.66 (m, 2H),4.02-3.91 (m, 6H), 3.843.74 (m, 5H), 3.71-3.62 (m, 5H), 3.51-3.48 (m,1H), 3.37-3.25 (m, 2H), 3.17 (br s, 2H), 2.85 (dd, J=14.5, 6.0 Hz, 1H),2.60 (m, 7H), 2.39-2.36, (m, 1H), 2.08-1.94 (m, 4H), 1.68-1.49 (m, 6H),1.33-1.28 (m, 1H), 0.96 (t, J=7.5 Hz, 3H), 0.85 (t, J=7.0 Hz, 3H); ESIMS m/z 881 [M+H]⁺.

Example 97 Preparation of 12′-Bromoanhydrovinblastine

To an ice cold solution of 12′-bromovinblastine (0.1 g, 0,09 mmol) inDMF (1 mL) was added thionyl chloride (0.04 mL, 0.45 mmol) and themixture stirred for 2 hours. The reaction mixture was diluted with 5%LiCl (aq) and extracted with dichloromethane (3×). The organic layer wasconcentrated under reduced pressure and the residue dried under highvacuum. Purification by column chromatography (silica, 94:4CHCl_(3/)MeOH) followed by reversed phase chromatography (C18,acetonitrile/water, 0.05% concentrated ammonium hydroxide) gave12′-bromoanhydrovinblastine (26 mg, 26%): ¹H NMR (300 MHz, CDCl₃) δ 7.62(d, J=1 Hz, 1H), 7.22 (dd, J=9, 1 Hz, 1H), 6.98 (d, J=8 Hz, 1H), 6.49(s, 1H), 6.11 (s, 1H), 5.86 (dd, J=10, 4 Hz, 1H), 5.45 (m, 1H), 5.45 (s,1H), 5.30 (d, J=10 Hz, 1H), 3.81 (s, 3H), 3.81 (m, 2H), 3.79 (s, 3H),3.73 (s, 1H), 3.64 (s, 3H), 3.53 (m, 1H), 3.45-3.05 (m, 4H), 3.00 (m,2H), 2.80 (m, 2H), 2.71 (s, 3H), 2.62 (s, 1H), 2.42 (m, 2H), 2.13 (m,1H), 2.11 (s, 3H), 2.03 (s, 1H), 1.93 (q, J=7 Hz, 2H), 1.76 (obs m),1.21 (m, 2H), 0.9 (t, J=7Hz, 3H), 0.77 (t, J=7Hz, 3H); ESI MS m/z 871,873 [M+H]⁺.

Example 98 Preparation of 12′-Iodoanhydrovinblastine

12′-iodoanhydrovinblastine was prepared from 12′-iodovinblastinefollowing the procedure described in Example 98 and was used withoutpurification, yield (0.25 g, quant.): ¹H NMR (free base, 300 MHz, MeOD)δ 7.34 (s, 1H), 7.33 (d, J=8 Hz, 1H), 7.01 (d, J=8 Hz, 1H), 6.56 (s,1H), 6.33 (s, 1H), 5.85 (dd, J=10, 4 Hz, 1H), 5.49 (m, 1H), 5.35 (s,1H), 5.30 (m, 1H), 4.61 (m, 1H), 3.90-3.55 (m, 7H), 3.83 (s, 3H), 3.76(s, 3H), 3.65 (s, 3H), 3.60 (s, 1H), 3.45 (d, J=16 Hz, 1H), 3.10-3.37(m, 4H), 2.88 (dd, J=14, 5 Hz, 1H), 2.73 (s, 3H), 2.47 (d, J =16 Hz,1H), 2.32 (m, 1H), 2.02 (s, 3H), 2.00 (m, 1H), 1.45-1.80 (m, 6H), 1.02(t, J=7 Hz, 3H), 0.72 (t, J=7 Hz, 3H); ESI m/z 919 [M+H]⁺.

Example 99 Preparation of 12′-Bromoanhydrovincristine

12′-bromoanhydrovincristine was prepared from 12′-bromovincristine andpurified following the procedure described in Example 98, yield (32 mg,23%). ¹H NMR (300 MHz, MeOD) δ 8.89 (s, 1H), 7.52 (s, 1H), 7.18 (s, 1H),7.10 (m, 2H), 6.82 (s, 1H), 5.82 (dd, J=10, 4 Hz, 1H), 5.40 (m, 1H),5.43-5.30 (m, 2H), 5.07 (s, 1H), 4.52 (s, 1H), 3.85 (s, 3H), 3.62 (t,J=10 Hz, 1H), 3.61 (s, 3H), 3.55 (s, 3H), 3.40-3.08 (m), 3.08-2.87 (m,3H), 2.80 (d, J=16 Hz, 1H), 2.56 (m, 1H), 2.38 (m, 2H), 1.93 (s, 3H),1.89 (q, J=8 Hz, 2H), 1.70 (m, 2H), 1.42 (m, 2H), 1.22 (m, 2H), 0.94 (t,J=8 Hz, 3H), 0.64 (t, J=7 Hz, 3H); ESI m/z 885, 887 [M+H]⁺.

Example 100 Description of Biological Assays

A. HeLa GI₅₀ Determinations

Growth inhibition (GI₅₀) values were measured on the human cervicalcarcinoma cell line, HeLa S-3, which were selected for growth onplastic. The HeLa cell assay was based on the description of Skehan etal., J. Natl. Cancer Inst., 82:1107-12 (1990), which is herebyincorporated by reference in its entirety. HeLa cells were plated at2×10⁴ cells/well in 96 well plates. One day later, a control plate wasfixed by the addition of TCA to 5%. After five rinses with tap water,the plate was air-dried and stored at 4° C. Test compounds were added tothe remaining plates at 10-fold dilutions. Two days later, all plateswere fixed as described above. Cells were then stained by the additionof 100 μL per well of 0.4% sulforhodamine B (SRB) in 1% acetic acid for30 min at 4° C. Wells were then quickly rinsed 5× with 1% acetic acidand allowed to air dry. The SRB was then solubilized by the addition of100 μL per well of unbuffered 10 mM Tris base. Dye was quantified bymeasuring absorbance at 490 nm on a Molecular Devices microplate reader.Growth inhibition was calculated according to the following equation:GI=100×(T−T₀)/(C−T₀), where the optical density (OD) of the test wellafter 2 days of treatment was T, the OD of the wells in the controlplate on day 0 was T₀ and C was the OD of untreated wells. Plots ofpercent growth inhibition versus inhibitor concentration were used todetermine the GI₅₀.

B. MCF-7 GI₅₀ Determinations

Growth inhibition (GI₅₀) values were measured on the human breastcarcinoma line, MCF-7. MCF-7 cells were plated at 2×10⁴ cells/well in 96well plates and grown for 24 hours in drug free media. On day 2, testcompounds were added to the plates at 10-fold dilutions. Four dayslater, cells were fixed by the addition of glutaraldehyde to 0.75%.After 30 min, the fixed cells were extensively rinsed with distilledwater and dried at room temperature for one hour. The cells were thenstained with a 0.2% crystal violet solution for one hour at roomtemperature. Unbound stain was removed by ten rinses with tap water andplates were allowed to air dry for 30 min. The crystal violet was thensolubilized by the addition of 10% acetic acid for 15 min and quantifiedby measuring absorbance at 570 nm on a Molecular Devices microplatereader. Growth inhibition was calculated according to the followingequation: GI=100×(T/T₀), where the optical density (OD) of the test wellafter 4 days of treatment was T, the OD of the wells in the controlplate on day 0 was T₀. Plots of percent growth inhibition versusinhibitor concentration were used to determine the GI₅₀.

TABLE 3 Growth Inhibition (GI₅₀) of HeLa Cells for Compounds of theCurrent Invention. HeLa Cells MCF-7 Cells Example GI₅₀ (nM) GI₅₀ (nM) 550 30 6 400 300 7 400 100 8 ND ND 9 300 500 10 250 95 11 300 300 12 200300 13 >1000 600 14 300 300 15 200 100 16 300 40 17 ND ND 18 300 30019 >1000 800 20 500 >1000 21 300 300 22 300 300 23 40 50 24 300 400 25 39 26 165 70 27 200 300 28 100 300 29 515 515 30 65 45 31 600 300 32 3530 33 20 30 35 200 60 36 30 50 37 100 200 38 100 200 39 25 25 40 6 8 412 3 42 3 3 43 0.6 2 44 70 100 45 50 200 46 200 500 47 20 40 48 30 40 498 30 50 20 40 52 200 300 53 20 30 54 100 50 55 19 6 56 30 30 57 300 30058 200 100 59 300 300 60 10 20 61 3 3 62 10 30 63 40 60 64 900 >1000 653 3 66 10 4 67 20 30 68 30 200 69 600 >1000 70 200 100 71 200 100 72 4050 73 200 300 74 30 40 75 0.3 0.3 76 1 3 77 0.3 0.6 78 5 10 79 700 >100080 200 200 81 30 50 82 300 600 83 300 500 84 30 500 85 20 30 86 30 50 8750 60 90 30 30 91 60 200 92 30 30 93 20 30 94 30 50 95 20 30 96 300 60097 30 50 98 50 305 99 4 1D. NCI Sixty Cell Line Data

The following data in Table 4 summarize the growth inhibition propertiesof several compounds of the present invention against 60-humantransformed cell lines. These data were cooperatively obtained at theNational Cancer Institute in their 60-cell line growth inhibition assayaccording to published procedures (Boyd, M. R., “Anticancer DrugDevelopment Guide,” Preclinical Screening, Clinical Trials, andApproval; Teicher, B. Ed.; Humana Press; Totowa, N.J., 23-42 (1997),which is hereby incorporated by reference).

TABLE 4 In Vitro Growth Inhibition (GI₅₀) of NCI Human Transformed CellLines of Several Compounds of the Current Invention. 17 GI₅₀ 19 GI₅₀ 29GI₅₀ 97 GI₅₀ Cancer Type Cell Line (nM) (nM) (nM) (nM) Breast BT-549 <101890 — — Breast HS 578T <10 1850 628 <10 Breast MCF7 <10 396 10.5 <10Breast MDA-MB- <10 2020 <10 <10 231/ATCC Breast MDA-MB-435 <10 60.5 34.3<10 Breast NCI/ADR-RES 2090 1440 18100 77.9 Breast T-47D — 4750 — 19400CNS SF-268 — 731 901 <10 CNS SF-295 <10 293 568 <10 CNS SF-539 <10 179021 <10 CNS SNB-19 <10 1790 <10 <10 CNS SNB-75 <10 530 — 12200 CNS U251<10 9970 <10 <10 Colon COLO 205 <10 1190 <10 <10 Colon HCC-2998 <10 182<10 <10 Colon HCT-116 <10 300 <10 <10 Colon HCT-15 44 8350 274 <10 ColonHT29 <10 423 <10 <10 Colon KM12 <10 206 212 <10 Colon SW-620 <10 17701300 <10 Leukemia CCRF-CEM — 263 28.8 <10 Leukemia HL-60(TB) — — 244 <10Leukemia K-562 53.1 — <10 <10 Leukemia MOLT-4 <10 196 11 <10 LeukemiaRPMI-8226 — 252 14.1 <10 Leukemia SR — — 396 — Melanoma LOX IMVI<10 >100000 <10 <10 Melanoma M14 <10 260 <10 <10 Melanoma MALME-3M <10254 — 1420 Melanoma SK-MEL-2 <10 2290 9770 — Melanoma SK-MEL-28 — 1270 —3310 Melanoma SK-MEL-5 <10 757 420 <10 Melanoma UACC-257 22400 4600019300 3950 Melanoma UACC-62 <10 2040 <10 <10 Non-Small Cell A549/ATCC16.9 37300 28.7 <10 Lung Non-Small Cell EKVX <10 2790 10800 10200 LungNon-Small Cell HOP-62 <10 571 <10 <10 Lung Non-Small Cell HOP-92 160 3359930 12200 Lung Non-Small Cell NCI-H226 — — <10 <10 Lung Non-Small CellNCI-H23 <10 541 — 325 Lung Non-Small Cell NCI-H322M <10 429 955 — LungNon-Small Cell NCI-H460 <10 353 176 <10 Lung Non-Small Cell NCI-H522 <101810 31.4 <10 Lung Ovarian IGROV1 — 2210 1710 <10 Ovarian OVCAR-3 <10207 59.7 <10 Ovarian OVCAR-4 15.3 1550 21.1 — Ovarian OVCAR-5 <10 1780 —12.4 Ovarian OVCAR-8 <10 32200 22.6 <10 Ovarian SK-OV-3 <10 1880 16.9<10 Prostate DU-145 <10 2460 15.2 <10 Prostate PC-3 <10 280 44.2 <10Renal 786-0 <10 889 <10 <10 Renal A498 <10 — — — Renal ACHN <10 2200093.7 64.1 Renal CAKI-1 73.4 557 1200 589 Renal RXF 393 <10 619 268 —Renal SN12C <10 2470 <10 <10 Renal TK-10 <10 313 3240 <10 Renal UO-3198.6 2200 — — Renal RPMI-8226 — 252 14.1 <10

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A compound of Formula (I) as follows:

where: R₁ is: alkyl; alkenyl; alkynyl; aryl; pyrazol-1-yl; pyridyl; thiophenyl; thiazolyl; piperidinyl; morpholinyl; pyrrolidinyl; azetidinyl; Cl; Br; F; CN; C(O)NR₅R₆; C(O)NHR₅; C(O)NH₂; C(O)NHNH₂; C(O)NR₅NH₂; C(O)NR₅NHR₆; C(O)NR₅NR₆R₇; C(O)NHNHR₅; C(O)NHNR₅R₆; C(O)NHOH; SO₂NHNH₂; SO₂NR₅NH₂; SO₂NR₅NHR₆; SO₂NR₅NR₆R₇; SO₂NHNHR₅; SO₂NHNR₅R₆; CO₂R₅; SR₅; SSR₅; SO₂NHR₅; SO₂NR₅R₆; B(OR₅)₂; CF₃; SH; SO₂NH₂; NH₂; NHR₅; NHSO₂R₅; NR₅R₆; NHCOR₅; NR₅COR₆; or NR₅SO₂R₆; R₂=alkyl or CH(O); R₃=hydrogen, alkyl, or C(O)R₅; R₄=hydrogen or C(O)R₅; R₅, R₆ and R₇ each are independently alkyl, alkenyl, alkynyl, or aryl; R₈=hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, acyl, or thioalkyl; R₉=OH and R₁₀=H or R₉ and R₁₀ together form a bridging double bond; R₅ and R₆ could form a ring or R₆ and R₇ could form a ring; X=OR₅, NR₅R₆, NHNH₂, NHNHC(O)R₅, OH, NHR₅, NH₂, or NHNHC(O)H; R₄ and X may be linked together with intervening atoms to form a ring; R₁ and R₈ may be linked together; or a pharmaceutically acceptable salt thereof, wherein the alkyl and alkenyl groups may be branched, straight, with the proviso, that when R₈=H, R₉=OH, and R₁₀=H, then R₁≠Br and the proviso that if R₁ is an unsubstituted aryl, then R₂≠CH₃.
 2. The compound according to claim 1, wherein R₃=acetyl.
 3. The compound according to claim 1, wherein R₄=hydrogen.
 4. The compound according to claim 1, wherein X=OMe.
 5. The compound according to claim 1, wherein R₃=acetyl, R₄=hydrogen, and X=OMe.
 6. The compound according to claim 1, wherein R₂=CH(O).
 7. The compound according to claim 1, wherein R₂=alkyl.
 8. A compound of the following chemical formula:


9. A compound of the following chemical formula:


10. A compound of the following chemical formula:


11. A compound of the following chemical formula:


12. A compound of the following chemical formula:


13. A compound of the following chemical formula:


14. A compound of the following chemical formula:


15. The compound according to claim 1, wherein the compound has the following chemical formula:


16. The compound according to claim 1, wherein the compound has the following chemical formula:


17. The compound according to claim 1, wherein the compound has the following chemical formula:


18. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


19. The compound according to claim 1, wherein the compound has the following chemical formula:


20. The compound according to claim 1, wherein the compound has the following chemical formula:


21. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


22. The compound according to claim 1, wherein the compound has the following chemical formula:


23. The compound according to claim 1, wherein the compound has the following chemical formula:


24. The compound according to claim 1, wherein the compound has the following chemical formula:


25. The compound according to claim 1, wherein the compound has the following chemical formula:


26. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


27. The compound according to claim 1, wherein the compound has the following chemical formula:


28. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


29. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


30. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


31. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


32. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


33. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


34. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


35. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


36. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


37. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


38. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


39. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


40. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


41. The compound according to claim 1, wherein the compound has the following chemical formula:


42. The compound according to claim 1, wherein the compound has the following chemical formula:


43. The compound according to claim 1, wherein the compound has the following chemical formula:


44. The compound according to claim 1, wherein the compound has the following chemical formula:


45. The compound according to claim 1, wherein the compound has the following chemical formula:


46. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


47. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


48. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


49. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


50. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


51. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


52. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


53. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


54. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


55. The compound according to claim 1, wherein the compound has the following chemical formula:


56. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


57. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


58. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


59. The compound according to claim 1, wherein the compound has the following chemical formula:


60. The compound according to claim 1, wherein the compound has the following chemical formula:


61. The compound according to claim 1, wherein the compound has the following chemical formula:


62. A compound having the following chemical formula:


63. A compound of the following chemical formula:


64. A compound of the following chemical formula:


65. The compound according to claim 1, wherein the compound has the following chemical formula:


66. The compound according to claim 1, wherein the compound has the following chemical formula:


67. The compound according to claim 1, wherein the compound has the following chemical formula:


68. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


69. The compound according to claim 1, wherein the compound has the following chemical formula:


70. A compound of the following chemical formula:


71. A compound of the following chemical formula:


72. The compound according to claim 1, wherein the compound has the following chemical formula:


73. A compound of the following chemical formula:


74. The compound according to claim 1, wherein the compound has the following chemical formula:


75. A compound of the following chemical formula:


76. The compound according to claim 1, wherein the compound has the following chemical formula:


77. The compound according to claim 1, wherein the compound has the following chemical formula:


78. The compound according to claim 1, wherein the compound has the following chemical formula:


79. The compound according to claim 1, wherein the compound has the following chemical formula:


80. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


81. The compound according to claim 1, wherein the compound has a substituted form of R₁ with the following chemical formula:


82. The compound according to claim 1, wherein the compound has the following chemical formula:


83. The compound according to claim 1, wherein the compound has the following chemical formula:


84. The compound according to claim 1, wherein the compound has the following chemical formula:


85. The compound according to claim 1, wherein the compound has the following chemical formula:


86. A compound of the following chemical formula:


87. A compound of the following chemical formula:


88. A compound of the following chemical formula:


89. A compound of the following chemical formula:


90. A compound of the following chemical formula:


91. A compound of the following chemical formula:


92. A compound of the following chemical formula:


93. The compound according to claim 1, wherein the compound has the following chemical formula:


94. The compound according to claim 1, wherein the compound has the following chemical formula:


95. A complex comprising 2 structures of Formula I, according to claim 1, joined together at their R₁ groups, wherein each R₁ is —S—.
 96. A process for preparation of a derivative product compound of Formula (I) as follows:

where: R₁ is: alkyl; alkenyl; alkynyl; aryl; pyrazol-1yl; pyridyl; thiophenyl; thiazolyl; piperidinyl; morpholinyl; pyrrolidinyl; azetidinyl; CN; CH(O); COR₅; C(O)NR₅R₆; C(O)NHR₅; C(O)NH₂; C(O)NHNH₂; C(O)NR₅NH₂; C(O)NR₅NHR₆; C(O)NHNHR₅; C(O)NHNR₅R₆; C(O)NHOH; SO₂NHNH₂; SO₂NR₅NH₂; SO₂NR₅NHR₆; SO₂NHNHR₅; SO₂NHNR₅R₆; CO₂R₅; SR₅; SSR₅; SO₂NHR₅; SO₂NR₅R₆; B(OR₅)₂; CF₃; SH; SO₂NH₂; NH₂; NHR₅; NHSO₂R₅; NR₅R₆; NHCOR₅; NR₅COR₆; or NR₅SO₂R₆; R₂=alkyl or CH(O); R₃=hydrogen, alkyl, or C(O)R₅; R₄=hydrogen or C(O)R₅; R₅ and R₆ each are independently alkyl, alkenyl, alkynyl, or aryl; R₈=hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, acyl, or thioalkyl; R₉=OH and R₁₀=H or R₉ and R₁₀ together form a bridging double bond; R₅ and R₆ can form a ring; X=OR₅, NR₅R₆, NHNH₂, NHNHC(O)R₅, OH, NHR₅, NH₂, or NHNHC(O)H; R₄ and X may be linked together with intervening atoms to form a ring; R₁ and R₈ may be linked together; or a pharmaceutically acceptable salt thereof, wherein the alkyl and alkenyl groups may be branched, straight, said process comprising: converting an intermediate compound of formula:

wherein Y₁ is a halogen and Y₂ is a halogen or hydrogen, under conditions effective to produce the product compound of Formula (I).
 97. The process of claim 96 further comprising: reacting a starting material compound of formula:

under conditions effective to form the intermediate compound.
 98. The process of claim 97, wherein the conditions effective to form the intermediate compound include using a halogenating agent selected from the group consisting of N-bromosuccinimide, N-iodo-succinimide, and iodine monochloride.
 99. The process of claim 97, wherein said converting comprises: reacting the intermediate compound with a palladium catalyst reagent to produce the product of Formula (I).
 100. The process of claim 99, wherein the palladium catalyst reagent is selected from the group consisting of palladium acetate, tris(dibenzylideneacetone)dipalladium(0), 1,1′-bis(diphenylphosphino)ferrocene-dichloropalladium(II), tetrakis(triphenylphosphine)palladium, and bis(triphenylphosphine) palladium(II)dichloride.
 101. A process for preparation of a product compound of Formula (I) as follows:

where: R₁ is halogen; R₂=alkyl or CH(O); R₃=hydrogen, alkyl, or C(O)R₅; R₄=hydrogen or C(O)R₅; R₅ and R₆ each are independently alkyl, alkenyl, alkynyl, or aryl; R₈=hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, acyl, or thioalkyl; R₉=OH and R₁₀=H or R₉ and R₁₀ together form a bridging double bond; R₅ and R₆ can form a ring; X=OR₅, NR₅R₆, NHNH₂, NHNHC(O)R₅, OH, NHR₅, NH₂, or NHNHC(O)H; R₄ and X may be linked together with intervening atoms to form a ring; R₁ and R₈ may be linked together; or a pharmaceutically acceptable salt thereof, wherein the alkyl and alkenyl groups may be branched or straight, with the proviso, that when R₈=H, R₉=OH, and R₁₀=H, then R₁≠Br or I, said process comprising: reacting a starting material compound of formula:

under conditions effective to form the product compound.
 102. The process of claim 101, wherein the conditions effective to form the intermediate compound include using a halogenating agent selected from the group consisting of N-bromosuccinimide, N-iodo-succinimide, and iodine monochloride.
 103. A composition of matter comprising the compound of claim 1 and one or more excipients.
 104. The composition according to claim 103, wherein R₃=acetyl.
 105. The composition according to claim 103, wherein R₄=hydrogen.
 106. The composition according to claim 103, wherein X=OMe.
 107. The composition according to claim 103, wherein R₃=acetyl, R₄=hydrogen, and X=OMe.
 108. The composition according to claim 103, wherein R₂=CH(O).
 109. The composition according to claim 103, wherein R₂=alkyl. 